Lesson 14: R for Biochemistry & Molecular Biology
Modesto
2022-12-14 (17:12 h)
Wellcome & Disclaimer
This site contains the materials for the Coding tools for Biochemistry & Molecular Biology (Herramientas de Programación para Bioquímica y Biología Molecular) course of fall 2022 in the Bachelor’s Degree in Biochemistry @UAM. This materials are the basis for GitHub-pages-based website that can be accessed here. Detailed academic information about the course contents, dates and assessment only can be found at the UAM Moodle site.
All this material is open access and it is shared under CC BY-NC license.
Working with sequences
Regular expressions as a shortcut to explore sequence
Biological data, like protein or nucleic acid sequences can be stored and explored in different formats, each following its own structure. As you can imagine, that facilitates the use of regular expressions in any programming language to access that data. The use of regular expressions in R is similar to other languages, particularly, to the use you learnt in Python. Anyway, I suggest that you have a look to the References below. However, for advanced manipulation of string object, I’d advice the package Strings.
In the code below, we are going to use some simple regular expressions to explore and analyze the data in a multifasta file containing the whole proteome of the bacteria Bacillus thuringiensis HER1410.
# read the text file with readLines()
proteome <- readLines("data/HER1410.fasta")
# number of sequences in the multifasta using grep
length(grep(proteome, pattern = ">"))## [1] 5807# extract of recombinases
grep("recombinase", proteome, value = TRUE)## [1] ">QKO37045.1 recombinase RecA (plasmid) [Bacillus thuringiensis]"
## [2] ">QKO36998.1 recombinase family protein (plasmid) [Bacillus thuringiensis]"
## [3] ">QKO36701.1 DDE-type integrase/transposase/recombinase (plasmid) [Bacillus thuringiensis]"
## [4] ">QKO36696.1 tyrosine recombinase XerS (plasmid) [Bacillus thuringiensis]"
## [5] ">QKO36060.1 tyrosine-type recombinase/integrase [Bacillus thuringiensis]"
## [6] ">QKO36057.1 tyrosine-type recombinase/integrase [Bacillus thuringiensis]"
## [7] ">QKO35589.1 recombinase family protein [Bacillus thuringiensis]"
## [8] ">QKO35551.1 tyrosine-type recombinase/integrase [Bacillus thuringiensis]"
## [9] ">QKO35280.1 recombinase family protein [Bacillus thuringiensis]"
## [10] ">QKO34873.1 site-specific tyrosine recombinase XerD [Bacillus thuringiensis]"
## [11] ">QKO34618.1 tyrosine recombinase XerC [Bacillus thuringiensis]"
## [12] ">QKO34567.1 recombinase RecA [Bacillus thuringiensis]"
## [13] ">QKO34482.1 recombinase family protein [Bacillus thuringiensis]"
## [14] ">QKO32507.1 recombinase RecQ [Bacillus thuringiensis]"
## [15] ">QKO32484.1 tyrosine-type recombinase/integrase [Bacillus thuringiensis]"# enzymes (aka '-ases')
head(grep("ase", proteome, value = TRUE))## [1] ">QKO37046.1 helicase DnaB (plasmid) [Bacillus x1]"
## [2] ">QKO37045.1 recombinase RecA (plasmid) [Bacillus thuringiensis]"
## [3] ">QKO37043.1 IS3 family transposase (plasmid) [Bacillus thuringiensis]"
## [4] ">QKO37040.1 serine acetyltransferase (plasmid) [Bacillus thuringiensis]"
## [5] ">QKO37039.1 O-antigen ligase family protein (plasmid) [Bacillus thuringiensis]"
## [6] ">QKO37026.1 HK97 family phage prohead protease (plasmid) [Bacillus thuringiensis]"head(grep("ase", proteome, value = TRUE, ignore.case = TRUE)) #lower- & uppercase## [1] ">QKO37046.1 helicase DnaB (plasmid) [Bacillus x1]"
## [2] ">QKO37045.1 recombinase RecA (plasmid) [Bacillus thuringiensis]"
## [3] ">QKO37043.1 IS3 family transposase (plasmid) [Bacillus thuringiensis]"
## [4] ">QKO37040.1 serine acetyltransferase (plasmid) [Bacillus thuringiensis]"
## [5] ">QKO37039.1 O-antigen ligase family protein (plasmid) [Bacillus thuringiensis]"
## [6] "SMSCDIDPTEIITRLTPLGTRIESKNEGATDASEARLTIESVNNGVPYIDHPSGIKEFGIQGKSITWDDV"# extract names
fastanames <- proteome[grep(proteome, pattern = ">")] #extract the names of the sequences
# remove some pattern
head(sub(">", "", grep("ase", proteome, value = TRUE, ignore.case = TRUE)),
20)## [1] "QKO37046.1 helicase DnaB (plasmid) [Bacillus x1]"
## [2] "QKO37045.1 recombinase RecA (plasmid) [Bacillus thuringiensis]"
## [3] "QKO37043.1 IS3 family transposase (plasmid) [Bacillus thuringiensis]"
## [4] "QKO37040.1 serine acetyltransferase (plasmid) [Bacillus thuringiensis]"
## [5] "QKO37039.1 O-antigen ligase family protein (plasmid) [Bacillus thuringiensis]"
## [6] "SMSCDIDPTEIITRLTPLGTRIESKNEGATDASEARLTIESVNNGVPYIDHPSGIKEFGIQGKSITWDDV"
## [7] "LEANTKQLEAEQKRLTSSFKLQNAELGANASEADKLELAQKQLRQQMEMTDRVVHNLEQQLSAAKRVYGE"
## [8] "GASENVLTLAKVYDVDLNEATRGAGQLMSQFGLSTQQTFDLLAAGAQAGLNYSDELFDNLSEYAPLFKQA"
## [9] "STVEYFSEAWSSFIEMMHEFFDPIGQFFSELWSGIVETASSWWSNLVTTASELWSQLTQAWQETWNTILT"
## [10] "FVVGKITEAEARSLGIEAGNGSVTVPEVIASEIITYAQEENLLRKYGSVHKTAGDMKYPVLVKKADANVR"
## [11] "QKO37026.1 HK97 family phage prohead protease (plasmid) [Bacillus thuringiensis]"
## [12] "QKO37024.1 terminase large subunit (plasmid) [Bacillus thuringiensis]"
## [13] "QKO37023.1 P27 family phage terminase small subunit (plasmid) [Bacillus thuringiensis]"
## [14] "QKO37022.1 HNH endonuclease (plasmid) [Bacillus thuringiensis]"
## [15] "QKO37016.1 nuclease (plasmid) [Bacillus thuringiensis]"
## [16] "QKO37013.1 DNA primase (plasmid) [Bacillus thuringiensis]"
## [17] "QKO37011.1 AAA family ATPase (plasmid) [Bacillus thuringiensis]"
## [18] "QKO37005.1 sigma-70 family RNA polymerase sigma factor (plasmid) [Bacillus thuringiensis]"
## [19] "QKO36998.1 recombinase family protein (plasmid) [Bacillus thuringiensis]"
## [20] "QKO36995.1 IS3 family transposase (plasmid) [Bacillus thuringiensis]"# regexpr returns the indices of the string where the match
# begins and the length of the match
(r <- regexpr("(.*)DNA(.*)", fastanames[1:200])) #find all the lines with 'DNA' (only 200 lines to don't make it too long)## [1] -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## [26] -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## [51] -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## [76] -1 -1 1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## [101] -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## [126] -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## [151] -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## [176] -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## attr(,"match.length")
## [1] -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## [26] -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 58 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## [51] -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## [76] -1 -1 95 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## [101] -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## [126] -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## [151] -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 68 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## [176] -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
## attr(,"index.type")
## [1] "chars"
## attr(,"useBytes")
## [1] TRUE# which ones?
(dna <- which(r > 0, arr.ind = TRUE))## [1] 37 78 165dna.length <- attr(r, "match.length")[dna]
# extract the results with a loop
nombres <- list()
for (i in 1:length(dna)) {
nombres[[i]] <- substr(fastanames[dna[i]], 1, 1 + dna.length[i])
}
head(nombres)## [[1]]
## [1] ">QKO37013.1 DNA primase (plasmid) [Bacillus thuringiensis]"
##
## [[2]]
## [1] ">QKO36972.1 DNA-3-methyladenine glycosylase 2 family protein (plasmid) [Bacillus thuringiensis]"
##
## [[3]]
## [1] ">QKO36885.1 DNA topoisomerase III (plasmid) [Bacillus thuringiensis]"Working with sequences as objects
The use or regular expressions is very convenient for some quick overview of the (multi)fasta files, but not for more specific analyses. Working with sequences can be facilitated with diverse packages, being seqinr and Biostrings the more widely used in the literature.
In the following example, we are reading the same protein multifasta and we will obtain basic statistics, subset and save fasta sequences using SeqinR (=seqinr).
NOTE: From this point on, we will show only the first 25 lines of the output of some code chunks (you will notice that the output starts and ends with …). Otherwise, this document is very long and difficult to follow. Run the code yourself to see the full returned output.
# reading fasta and playing with it
# install.packages('seqinr')
library(seqinr)
prots <- read.fasta("data/HER1410.fasta")
str(prots)...
## List of 5807
## $ QKO37049.1: 'SeqFastadna' chr [1:67] "m" "t" "l" "t" ...
## ..- attr(*, "name")= chr "QKO37049.1"
## ..- attr(*, "Annot")= chr ">QKO37049.1 hypothetical protein HBA75_30905 (plasmid) [Bacillus thuringiensis]"
## $ QKO37048.1: 'SeqFastadna' chr [1:142] "m" "k" "v" "l" ...
## ..- attr(*, "name")= chr "QKO37048.1"
## ..- attr(*, "Annot")= chr ">QKO37048.1 hypothetical protein HBA75_30875 (plasmid) [Bacillus thuringiensis]"
## $ QKO37047.1: 'SeqFastadna' chr [1:326] "m" "e" "k" "k" ...
## ..- attr(*, "name")= chr "QKO37047.1"
## ..- attr(*, "Annot")= chr ">QKO37047.1 cytosolic protein (plasmid) [Bacillus thuringiensis]"
## $ QKO37046.1: 'SeqFastadna' chr [1:146] "m" "e" "k" "q" ...
## ..- attr(*, "name")= chr "QKO37046.1"
## ..- attr(*, "Annot")= chr ">QKO37046.1 helicase DnaB (plasmid) [Bacillus x1]"
## $ QKO37045.1: 'SeqFastadna' chr [1:359] "m" "a" "a" "t" ...
## ..- attr(*, "name")= chr "QKO37045.1"
## ..- attr(*, "Annot")= chr ">QKO37045.1 recombinase RecA (plasmid) [Bacillus thuringiensis]"
## $ QKO37044.1: 'SeqFastadna' chr [1:110] "m" "s" "y" "d" ...
## ..- attr(*, "name")= chr "QKO37044.1"
## ..- attr(*, "Annot")= chr ">QKO37044.1 hypothetical protein HBA75_31045 (plasmid) [Bacillus thuringiensis]"
## $ QKO37043.1: 'SeqFastadna' chr [1:293] "m" "k" "a" "a" ...
## ..- attr(*, "name")= chr "QKO37043.1"
## ..- attr(*, "Annot")= chr ">QKO37043.1 IS3 family transposase (plasmid) [Bacillus thuringiensis]"
## $ QKO37042.1: 'SeqFastadna' chr [1:102] "m" "g" "k" "i" ...
## ..- attr(*, "name")= chr "QKO37042.1"
## ..- attr(*, "Annot")= chr ">QKO37042.1 helix-turn-helix domain-containing protein (plasmid) [Bacillus thuringiensis]"
...getName(prots)...
## [1] "QKO37049.1" "QKO37048.1" "QKO37047.1" "QKO37046.1" "QKO37045.1"
## [6] "QKO37044.1" "QKO37043.1" "QKO37042.1" "QKO37041.1" "QKO37040.1"
## [11] "QKO37039.1" "QKO37038.1" "QKO37037.1" "QKO37036.1" "QKO37035.1"
## [16] "QKO37034.1" "QKO37033.1" "QKO37032.1" "QKO37031.1" "QKO37030.1"
## [21] "QKO37029.1" "QKO37028.1" "QKO37027.1" "QKO37026.1" "QKO37025.1"
## [26] "QKO37024.1" "QKO37023.1" "QKO37022.1" "QKO37021.1" "QKO37020.1"
## [31] "QKO37019.1" "QKO37018.1" "QKO37017.1" "QKO37016.1" "QKO37015.1"
## [36] "QKO37014.1" "QKO37013.1" "QKO37012.1" "QKO37011.1" "QKO37010.1"
## [41] "QKO37009.1" "QKO37008.1" "QKO37007.1" "QKO37006.1" "QKO37005.1"
## [46] "QKO37004.1" "QKO37003.1" "QKO37002.1" "QKO37001.1" "QKO37000.1"
## [51] "QKO36999.1" "QKO36998.1" "QKO36997.1" "QKO36996.1" "QKO36995.1"
## [56] "QKO36994.1" "QKO36993.1" "QKO36992.1" "QKO36991.1" "QKO36990.1"
## [61] "QKO36989.1" "QKO36988.1" "QKO36987.1" "QKO36986.1" "QKO36985.1"
## [66] "QKO36984.1" "QKO36983.1" "QKO36982.1" "QKO36981.1" "QKO36980.1"
## [71] "QKO36979.1" "QKO36978.1" "QKO36977.1" "QKO36976.1" "QKO36975.1"
## [76] "QKO36974.1" "QKO36973.1" "QKO36972.1" "QKO36971.1" "QKO36970.1"
## [81] "QKO36969.1" "QKO36968.1" "QKO36967.1" "QKO36966.1" "QKO36965.1"
## [86] "QKO36964.1" "QKO36963.1" "QKO36962.1" "QKO36961.1" "QKO36960.1"
## [91] "QKO36959.1" "QKO36958.1" "QKO36957.1" "QKO36956.1" "QKO36955.1"
## [96] "QKO36954.1" "QKO36953.1" "QKO36952.1" "QKO36951.1" "QKO36950.1"
## [101] "QKO36949.1" "QKO36948.1" "QKO36947.1" "QKO36946.1" "QKO36945.1"
## [106] "QKO36944.1" "QKO36943.1" "QKO36942.1" "QKO36941.1" "QKO36940.1"
## [111] "QKO36939.1" "QKO36938.1" "QKO36937.1" "QKO36936.1" "QKO36935.1"
## [116] "QKO36934.1" "QKO36933.1" "QKO36932.1" "QKO36931.1" "QKO36930.1"
## [121] "QKO36929.1" "QKO36928.1" "QKO36927.1" "QKO36926.1" "QKO36925.1"
...prots[[1]]## [1] "m" "t" "l" "t" "l" "h" "n" "g" "d" "l" "n" "k" "l" "a" "r" "d" "t" "s" "q"
## [20] "d" "s" "i" "i" "l" "r" "v" "g" "e" "q" "e" "m" "v" "s" "l" "k" "s" "n" "g"
## [39] "d" "i" "y" "v" "k" "g" "k" "l" "v" "e" "n" "d" "k" "e" "v" "v" "d" "g" "m"
## [58] "r" "e" "l" "l" "m" "l" "s" "r" "k" "r"
## attr(,"name")
## [1] "QKO37049.1"
## attr(,"Annot")
## [1] ">QKO37049.1 hypothetical protein HBA75_30905 (plasmid) [Bacillus thuringiensis]"
## attr(,"class")
## [1] "SeqFastadna"seq <- getSequence(prots[[1]])
getName(prots[[1]])## [1] "QKO37049.1"paste(seq, collapse = "")## [1] "mtltlhngdlnklardtsqdsiilrvgeqemvslksngdiyvkgklvendkevvdgmrellmlsrkr"paste(prots[[1]], collapse = "")## [1] "mtltlhngdlnklardtsqdsiilrvgeqemvslksngdiyvkgklvendkevvdgmrellmlsrkr"aaa(toupper(seq))## [1] "Met" "Thr" "Leu" "Thr" "Leu" "His" "Asn" "Gly" "Asp" "Leu" "Asn" "Lys"
## [13] "Leu" "Ala" "Arg" "Asp" "Thr" "Ser" "Gln" "Asp" "Ser" "Ile" "Ile" "Leu"
## [25] "Arg" "Val" "Gly" "Glu" "Gln" "Glu" "Met" "Val" "Ser" "Leu" "Lys" "Ser"
## [37] "Asn" "Gly" "Asp" "Ile" "Tyr" "Val" "Lys" "Gly" "Lys" "Leu" "Val" "Glu"
## [49] "Asn" "Asp" "Lys" "Glu" "Val" "Val" "Asp" "Gly" "Met" "Arg" "Glu" "Leu"
## [61] "Leu" "Met" "Leu" "Ser" "Arg" "Lys" "Arg"aaa(toupper(unlist(seq)))## [1] "Met" "Thr" "Leu" "Thr" "Leu" "His" "Asn" "Gly" "Asp" "Leu" "Asn" "Lys"
## [13] "Leu" "Ala" "Arg" "Asp" "Thr" "Ser" "Gln" "Asp" "Ser" "Ile" "Ile" "Leu"
## [25] "Arg" "Val" "Gly" "Glu" "Gln" "Glu" "Met" "Val" "Ser" "Leu" "Lys" "Ser"
## [37] "Asn" "Gly" "Asp" "Ile" "Tyr" "Val" "Lys" "Gly" "Lys" "Leu" "Val" "Glu"
## [49] "Asn" "Asp" "Lys" "Glu" "Val" "Val" "Asp" "Gly" "Met" "Arg" "Glu" "Leu"
## [61] "Leu" "Met" "Leu" "Ser" "Arg" "Lys" "Arg"aaa(toupper(as.vector(seq)))## [1] "Met" "Thr" "Leu" "Thr" "Leu" "His" "Asn" "Gly" "Asp" "Leu" "Asn" "Lys"
## [13] "Leu" "Ala" "Arg" "Asp" "Thr" "Ser" "Gln" "Asp" "Ser" "Ile" "Ile" "Leu"
## [25] "Arg" "Val" "Gly" "Glu" "Gln" "Glu" "Met" "Val" "Ser" "Leu" "Lys" "Ser"
## [37] "Asn" "Gly" "Asp" "Ile" "Tyr" "Val" "Lys" "Gly" "Lys" "Leu" "Val" "Glu"
## [49] "Asn" "Asp" "Lys" "Glu" "Val" "Val" "Asp" "Gly" "Met" "Arg" "Glu" "Leu"
## [61] "Leu" "Met" "Leu" "Ser" "Arg" "Lys" "Arg"pmw(toupper(unlist(seq)))## [1] 7602.706(stats1 <- AAstat(toupper(seq), plot = T))
## $Compo
##
## * A C D E F G H I K L M N P Q R S T V W Y
## 0 1 0 6 5 0 5 1 3 6 10 4 4 0 2 5 5 3 6 0 1
##
## $Prop
## $Prop$Tiny
## [1] 0.2089552
##
## $Prop$Small
## [1] 0.4477612
##
## $Prop$Aliphatic
## [1] 0.2835821
##
## $Prop$Aromatic
## [1] 0.02985075
##
## $Prop$Non.polar
## [1] 0.4477612
##
## $Prop$Polar
## [1] 0.5522388
##
## $Prop$Charged
## [1] 0.3432836
##
## $Prop$Basic
## [1] 0.1791045
##
## $Prop$Acidic
## [1] 0.1641791
##
##
## $Pi
## [1] 6.557213barplot(stats1$Compo, bty = "7")
# cool color palette
library(RColorBrewer)
coul = colorRampPalette(brewer.pal(9, "Set1"))(20) #palette is expanded from 9 to 20 colors
barplot(stats1$Compo, col = coul, bty = "7")
# dotplot
grep("recombinase", getAnnot(prots))## [1] 5 52 349 354 990 993 1461 1499 1770 2177 2432 2483 2568 4543 4566dotPlot(prots[[5]], prots[[2483]], wsize = 3, nmatch = 2)
dotPlot(prots[[2177]], prots[[2432]], wsize = 3, nmatch = 2)
dotPlot(prots[[2177]], prots[[2432]], col = c("floralwhite",
"dodgerblue"), wsize = 6, nmatch = 3, xlab = getName(prots[[2177]]),
ylab = getName(prots[[2432]]))
# extract the recombinases to a new file
recs <- prots[grep("recombinase", getAnnot(prots))]
# save to a file
write.fasta(recs, names = names(recs), file.out = "data/recombinases.faa")As you noticed in the code above, the function
read.fasta() can read any (multi)fasta file containing a
protein or nucleic acid sequence and create a list of lists. Thus, if
you want to extract a sequence as a single element, you need to extract
if from the list with unlist() or convert it to a vector
with as.vector().
Once you read the fasta, you can extract the headline name with
getName() or the sequence with getSequence().
Additionally, you can obtain and plot basic stats or compare sequences
using dotPlot(). Moreover, if you extract a subset of
sequences, you can use write.fasta() to save those
sequences in a new fasta file.
Finally, as in the example below, you can also play with a nucleotide
sequence and obtain some useful data, like the GC()
content, the encoded proteins or the reverse complementary sequence with
rev() and comp(), respectively.
# now the nt sequence
plasmid_nt <- read.fasta("data/HER1410nt.fasta")
count(unlist(plasmid_nt), 3)##
## aaa aac aag aat aca acc acg act aga agc agg agt ata atc atg att
## 2150 705 1089 1420 715 253 321 484 900 437 525 601 992 589 955 1354
## caa cac cag cat cca ccc ccg cct cga cgc cgg cgt cta ctc ctg ctt
## 848 248 455 548 325 85 126 235 297 126 190 308 448 200 360 569
## gaa gac gag gat gca gcc gcg gct gga ggc ggg ggt gta gtc gtg gtt
## 1273 273 418 829 467 128 212 384 641 224 292 516 620 230 484 755
## taa tac tag tat tca tcc tcg tct tga tgc tgg tgt tta ttc ttg ttt
## 1093 547 501 1093 592 305 262 475 955 404 666 663 1174 615 889 1310count(unlist(plasmid_nt), 5)##
## aaaaa aaaac aaaag aaaat aaaca aaacc aaacg aaact aaaga aaagc aaagg aaagt aaata
## 307 110 200 221 138 31 58 45 189 77 105 86 133
## aaatc aaatg aaatt aacaa aacac aacag aacat aacca aaccc aaccg aacct aacga aacgc
## 109 170 171 144 25 64 73 34 14 15 24 42 13
## aacgg aacgt aacta aactc aactg aactt aagaa aagac aagag aagat aagca aagcc aagcg
## 28 45 48 24 46 66 199 38 70 104 71 20 38
## aagct aagga aaggc aaggg aaggt aagta aagtc aagtg aagtt aataa aatac aatag aatat
## 61 97 33 40 70 96 13 55 84 112 80 52 116
## aatca aatcc aatcg aatct aatga aatgc aatgg aatgt aatta aattc aattg aattt acaaa
## 81 41 35 54 130 51 95 83 162 77 128 123 109
## acaac acaag acaat acaca acacc acacg acact acaga acagc acagg acagt acata acatc
## 54 63 67 27 18 20 24 52 34 39 44 37 27
## acatg acatt accaa accac accag accat accca acccc acccg accct accga accgc accgg
## 36 64 38 13 28 20 14 2 3 14 13 10 9
## accgt accta acctc acctg acctt acgaa acgac acgag acgat acgca acgcc acgcg acgct
## 16 19 5 20 29 45 8 14 35 9 6 2 23
## acgga acggc acggg acggt acgta acgtc acgtg acgtt actaa actac actag actat actca
## 32 13 8 21 35 10 27 33 39 24 20 44 30
## actcc actcg actct actga actgc actgg actgt actta acttc acttg acttt agaaa agaac
## 7 10 14 44 10 27 36 49 28 41 61 171 51
## agaag agaat agaca agacc agacg agact agaga agagc agagg agagt agata agatc agatg
## 109 84 35 16 19 13 54 15 39 39 85 19 75
## agatt agcaa agcac agcag agcat agcca agccc agccg agcct agcga agcgc agcgg agcgt
## 76 89 12 28 37 12 7 12 20 33 17 20 26
## agcta agctc agctg agctt aggaa aggac aggag aggat aggca aggcc aggcg aggct aggga
## 35 11 41 37 72 26 46 48 24 1 19 21 38
## agggc agggg agggt aggta aggtc aggtg aggtt agtaa agtac agtag agtat agtca agtcc
## 17 23 23 51 20 59 37 73 21 28 65 28 13
## agtcg agtct agtga agtgc agtgg agtgt agtta agttc agttg agttt ataaa ataac ataag
## 9 16 42 18 35 26 60 35 63 69 133 52 53
## ataat ataca atacc atacg atact ataga atagc atagg atagt atata atatc atatg atatt
## 84 79 29 40 46 46 25 20 36 79 74 77 119
## atcaa atcac atcag atcat atcca atccc atccg atcct atcga atcgc atcgg atcgt atcta
## 94 32 37 63 50 8 16 39 40 11 17 32 59
## atctc atctg atctt atgaa atgac atgag atgat atgca atgcc atgcg atgct atgga atggc
## 18 25 48 184 36 36 94 65 17 20 48 80 35
## atggg atggt atgta atgtc atgtg atgtt attaa attac attag attat attca attcc attcg
## 47 69 73 29 34 88 114 92 85 100 83 36 45
## attct attga attgc attgg attgt attta atttc atttg atttt caaaa caaac caaag caaat
## 50 111 43 91 76 153 60 89 126 133 45 57 99
## caaca caacc caacg caact caaga caagc caagg caagt caata caatc caatg caatt cacaa
## 44 11 18 42 59 32 32 47 63 25 38 103 39
## cacac cacag cacat cacca caccc caccg cacct cacga cacgc cacgg cacgt cacta cactc
## 12 26 22 14 3 10 17 14 7 11 11 13 8
## cactg cactt cagaa cagac cagag cagat cagca cagcc cagcg cagct cagga caggc caggg
## 12 29 52 15 28 46 39 10 26 27 29 13 19
## caggt cagta cagtc cagtg cagtt cataa catac catag catat catca catcc catcg catct
## 35 34 16 14 52 37 26 7 63 37 15 13 29
## catga catgc catgg catgt catta cattc cattg cattt ccaaa ccaac ccaag ccaat ccaca
## 38 15 21 18 68 35 46 80 46 14 28 35 27
## ccacc ccacg ccact ccaga ccagc ccagg ccagt ccata ccatc ccatg ccatt cccaa cccac
## 5 6 4 24 17 16 21 18 16 9 39 14 3
## cccag cccat cccca ccccc ccccg cccct cccga cccgc cccgg cccgt cccta ccctc ccctg
## 7 11 6 4 1 5 3 1 0 2 13 2 5
## ccctt ccgaa ccgac ccgag ccgat ccgca ccgcc ccgcg ccgct ccgga ccggc ccggg ccggt
## 8 19 6 2 8 7 6 3 6 9 3 2 7
## ccgta ccgtc ccgtg ccgtt cctaa cctac cctag cctat cctca cctcc cctcg cctct cctga
## 11 7 10 20 23 6 6 28 7 5 3 6 21
## cctgc cctgg cctgt cctta ccttc ccttg ccttt cgaaa cgaac cgaag cgaat cgaca cgacc
## 6 18 11 27 16 13 39 50 12 27 32 10 6
## cgacg cgact cgaga cgagc cgagg cgagt cgata cgatc cgatg cgatt cgcaa cgcac cgcag
## 3 14 10 7 5 9 31 9 38 34 18 7 15
## cgcat cgcca cgccc cgccg cgcct cgcga cgcgc cgcgg cgcgt cgcta cgctc cgctg cgctt
## 13 9 0 3 4 3 1 4 3 12 5 12 16
## cggaa cggac cggag cggat cggca cggcc cggcg cggct cggga cgggc cgggg cgggt cggta
## 36 9 18 23 9 5 3 11 9 2 3 7 23
## cggtc cggtg cggtt cgtaa cgtac cgtag cgtat cgtca cgtcc cgtcg cgtct cgtga cgtgc
## 2 13 17 32 16 5 35 14 5 6 13 24 12
## cgtgg cgtgt cgtta cgttc cgttg cgttt ctaaa ctaac ctaag ctaat ctaca ctacc ctacg
## 17 20 31 21 19 38 74 23 19 42 24 4 13
## ctact ctaga ctagc ctagg ctagt ctata ctatc ctatg ctatt ctcaa ctcac ctcag ctcat
## 26 24 5 11 20 42 30 33 58 29 9 12 24
## ctcca ctccc ctccg ctcct ctcga ctcgc ctcgg ctcgt ctcta ctctc ctctg ctctt ctgaa
## 13 4 6 11 9 5 4 14 18 10 5 27 76
## ctgac ctgag ctgat ctgca ctgcc ctgcg ctgct ctgga ctggc ctggg ctggt ctgta ctgtc
## 9 13 41 20 3 2 17 24 12 13 31 27 11
## ctgtg ctgtt cttaa cttac cttag cttat cttca cttcc cttcg cttct cttga cttgc cttgg
## 22 39 51 22 29 54 30 16 5 35 45 21 32
## cttgt cttta ctttc ctttg ctttt gaaaa gaaac gaaag gaaat gaaca gaacc gaacg gaact
## 30 70 27 33 69 205 71 92 140 68 23 28 38
## gaaga gaagc gaagg gaagt gaata gaatc gaatg gaatt gacaa gacac gacag gacat gacca
## 124 60 60 67 72 43 76 106 34 13 22 29 16
## gaccc gaccg gacct gacga gacgc gacgg gacgt gacta gactc gactg gactt gagaa gagac
## 5 12 14 22 6 11 16 24 11 14 24 74 15
## gagag gagat gagca gagcc gagcg gagct gagga gaggc gaggg gaggt gagta gagtc gagtg
## 23 39 21 14 12 13 37 8 21 30 23 20 25
## gagtt gataa gatac gatag gatat gatca gatcc gatcg gatct gatga gatgc gatgg gatgt
## 43 78 38 37 86 22 18 14 16 103 44 50 62
## gatta gattc gattg gattt gcaaa gcaac gcaag gcaat gcaca gcacc gcacg gcact gcaga
## 62 43 57 99 82 19 41 62 14 6 10 14 32
## gcagc gcagg gcagt gcata gcatc gcatg gcatt gccaa gccac gccag gccat gccca gcccc
## 31 23 15 27 16 21 54 18 3 14 15 5 3
## gcccg gccct gccga gccgc gccgg gccgt gccta gcctc gcctg gcctt gcgaa gcgac gcgag
## 1 1 6 3 5 11 10 4 11 18 24 10 7
## gcgat gcgca gcgcc gcgcg gcgct gcgga gcggc gcggg gcggt gcgta gcgtc gcgtg gcgtt
## 27 22 1 3 4 26 5 4 16 15 8 16 24
## gctaa gctac gctag gctat gctca gctcc gctcg gctct gctga gctgc gctgg gctgt gctta
## 38 15 17 38 15 11 11 15 51 22 18 24 31
## gcttc gcttg gcttt ggaaa ggaac ggaag ggaat ggaca ggacc ggacg ggact ggaga ggagc
## 15 26 36 105 44 47 69 25 10 18 19 53 19
## ggagg ggagt ggata ggatc ggatg ggatt ggcaa ggcac ggcag ggcat ggcca ggccc ggccg
## 28 28 49 13 46 68 32 6 20 26 14 2 3
## ggcct ggcga ggcgc ggcgg ggcgt ggcta ggctc ggctg ggctt gggaa gggac gggag gggat
## 6 12 8 12 18 17 11 17 20 50 12 16 35
## gggca gggcc gggcg gggct gggga ggggc ggggg ggggt gggta gggtc gggtg gggtt ggtaa
## 19 5 10 14 17 15 13 13 20 4 21 28 66
## ggtac ggtag ggtat ggtca ggtcc ggtcg ggtct ggtga ggtgc ggtgg ggtgt ggtta ggttc
## 28 18 40 18 6 15 14 69 35 29 34 32 28
## ggttg ggttt gtaaa gtaac gtaag gtaat gtaca gtacc gtacg gtact gtaga gtagc gtagg
## 28 56 99 37 29 73 39 12 14 21 36 15 18
## gtagt gtata gtatc gtatg gtatt gtcaa gtcac gtcag gtcat gtcca gtccc gtccg gtcct
## 19 38 50 50 70 29 9 15 27 23 2 6 11
## gtcga gtcgc gtcgg gtcgt gtcta gtctc gtctg gtctt gtgaa gtgac gtgag gtgat gtgca
## 16 5 9 12 16 10 14 26 73 15 26 58 30
## gtgcc gtgcg gtgct gtgga gtggc gtggg gtggt gtgta gtgtc gtgtg gtgtt gttaa gttac
## 5 8 28 72 13 16 31 30 16 15 48 64 29
## gttag gttat gttca gttcc gttcg gttct gttga gttgc gttgg gttgt gttta gtttc gtttg
## 28 72 42 29 19 42 50 34 39 55 64 44 51
## gtttt taaaa taaac taaag taaat taaca taacc taacg taact taaga taagc taagg taagt
## 93 193 46 108 123 56 22 24 59 39 21 43 48
## taata taatc taatg taatt tacaa tacac tacag tacat tacca taccc taccg tacct tacga
## 92 34 75 110 76 39 57 40 35 11 11 18 24
## tacgc tacgg tacgt tacta tactc tactg tactt tagaa tagac tagag tagat tagca tagcc
## 14 24 33 42 18 45 60 90 15 26 66 35 7
## tagcg tagct tagga taggc taggg taggt tagta tagtc tagtg tagtt tataa tatac tatag
## 20 23 29 11 21 32 34 17 27 48 95 50 31
## tatat tatca tatcc tatcg tatct tatga tatgc tatgg tatgt tatta tattc tattg tattt
## 84 86 39 38 51 79 40 65 61 99 59 90 126
## tcaaa tcaac tcaag tcaat tcaca tcacc tcacg tcact tcaga tcagc tcagg tcagt tcata
## 97 28 38 65 31 15 7 20 33 20 18 36 51
## tcatc tcatg tcatt tccaa tccac tccag tccat tccca tcccc tcccg tccct tccga tccgc
## 35 26 72 53 23 29 36 10 7 1 8 13 8
## tccgg tccgt tccta tcctc tcctg tcctt tcgaa tcgac tcgag tcgat tcgca tcgcc tcgcg
## 7 19 21 10 20 40 33 9 8 42 15 3 3
## tcgct tcgga tcggc tcggg tcggt tcgta tcgtc tcgtg tcgtt tctaa tctac tctag tctat
## 13 19 7 7 11 27 13 20 32 58 22 17 53
## tctca tctcc tctcg tctct tctga tctgc tctgg tctgt tctta tcttc tcttg tcttt tgaaa
## 22 11 8 25 23 4 17 28 49 27 48 63 182
## tgaac tgaag tgaat tgaca tgacc tgacg tgact tgaga tgagc tgagg tgagt tgata tgatc
## 50 128 112 28 15 15 27 34 19 24 35 74 29
## tgatg tgatt tgcaa tgcac tgcag tgcat tgcca tgccc tgccg tgcct tgcga tgcgc tgcgg
## 100 83 65 19 38 42 15 1 7 13 20 4 15
## tgcgt tgcta tgctc tgctg tgctt tggaa tggac tggag tggat tggca tggcc tggcg tggct
## 16 44 25 45 35 107 25 48 70 32 14 18 19
## tggga tgggc tgggg tgggt tggta tggtc tggtg tggtt tgtaa tgtac tgtag tgtat tgtca
## 49 14 19 30 58 27 74 62 67 21 37 68 20
## tgtcc tgtcg tgtct tgtga tgtgc tgtgg tgtgt tgtta tgttc tgttg tgttt ttaaa ttaac
## 18 12 23 37 6 51 29 70 48 68 88 164 49
## ttaag ttaat ttaca ttacc ttacg ttact ttaga ttagc ttagg ttagt ttata ttatc ttatg
## 50 112 70 30 28 72 91 40 44 51 101 60 85
## ttatt ttcaa ttcac ttcag ttcat ttcca ttccc ttccg ttcct ttcga ttcgc ttcgg ttcgt
## 127 76 23 43 70 55 12 19 30 27 13 14 34
## ttcta ttctc ttctg ttctt ttgaa ttgac ttgag ttgat ttgca ttgcc ttgcg ttgct ttgga
## 57 28 28 86 139 25 37 93 49 11 25 56 74
## ttggc ttggg ttggt ttgta ttgtc ttgtg ttgtt tttaa tttac tttag tttat tttca tttcc
## 23 36 90 63 17 52 99 146 57 84 147 57 35
## tttcg tttct tttga tttgc tttgg tttgt tttta ttttc ttttg ttttt
## 19 72 88 43 61 70 147 52 89 143uco(plasmid_nt[[1]], frame = 0)##
## aaa aac aag aat aca acc acg act aga agc agg agt ata atc atg att caa cac cag cat
## 722 239 389 463 238 84 114 144 286 145 148 192 318 190 328 464 291 88 156 187
## cca ccc ccg cct cga cgc cgg cgt cta ctc ctg ctt gaa gac gag gat gca gcc gcg gct
## 125 22 49 94 94 40 62 90 148 73 158 197 374 82 145 267 125 30 75 130
## gga ggc ggg ggt gta gtc gtg gtt taa tac tag tat tca tcc tcg tct tga tgc tgg tgt
## 241 78 96 184 220 78 181 249 408 194 181 327 178 88 88 155 341 118 185 223
## tta ttc ttg ttt
## 404 206 313 414uco(plasmid_nt[[1]], frame = 2)##
## aaa aac aag aat aca acc acg act aga agc agg agt ata atc atg att caa cac cag cat
## 720 234 340 462 233 89 93 175 283 131 182 225 376 182 328 460 300 82 152 177
## cca ccc ccg cct cga cgc cgg cgt cta ctc ctg ctt gaa gac gag gat gca gcc gcg gct
## 96 37 39 85 97 34 61 117 139 54 96 189 461 97 135 270 183 53 74 140
## gga ggc ggg ggt gta gtc gtg gtt taa tac tag tat tca tcc tcg tct tga tgc tgg tgt
## 215 66 109 193 211 73 144 244 332 167 144 391 210 111 80 177 273 129 247 192
## tta ttc ttg ttt
## 384 200 299 444translate(plasmid_nt[[1]])...
## [1] "V" "W" "S" "I" "K" "H" "N" "L" "T" "T" "S" "N" "E" "Y" "S" "*" "A" "A"
## [19] "I" "I" "L" "F" "*" "N" "L" "*" "P" "L" "L" "L" "V" "Q" "L" "D" "S" "Y"
## [37] "L" "H" "E" "Q" "V" "L" "*" "L" "R" "L" "*" "W" "K" "I" "Q" "L" "H" "L"
## [55] "L" "L" "L" "S" "*" "I" "R" "C" "L" "N" "C" "T" "I" "H" "L" "S" "T" "D"
## [73] "L" "K" "V" "L" "L" "F" "Q" "V" "K" "R" "L" "Y" "Q" "L" "Y" "*" "P" "V"
## [91] "V" "F" "E" "L" "C" "S" "L" "L" "G" "L" "G" "L" "T" "Y" "P" "F" "Y" "I"
## [109] "R" "S" "*" "E" "Y" "L" "L" "L" "*" "T" "D" "I" "L" "N" "V" "L" "S" "Y"
## [127] "L" "G" "F" "C" "Y" "I" "V" "L" "V" "N" "Y" "L" "T" "L" "L" "V" "S" "L"
## [145] "Q" "Y" "H" "Q" "D" "Q" "R" "W" "R" "L" "P" "R" "G" "L" "E" "I" "L" "F"
## [163] "Q" "L" "H" "P" "L" "E" "L" "E" "A" "Y" "R" "M" "F" "L" "Y" "D" "V" "L"
## [181] "S" "Y" "T" "Y" "S" "S" "L" "Q" "F" "Y" "Y" "G" "F" "L" "L" "V" "Y" "F"
## [199] "P" "K" "N" "A" "F" "R" "I" "W" "R" "T" "L" "K" "H" "*" "R" "I" "R" "K"
## [217] "N" "Y" "A" "I" "*" "L" "C" "L" "S" "F" "Y" "Y" "L" "V" "L" "V" "V" "Q"
## [235] "Y" "T" "K" "Y" "L" "Y" "Q" "*" "K" "F" "R" "N" "P" "F" "E" "Q" "C" "L"
## [253] "L" "E" "C" "R" "L" "P" "S" "G" "F" "F" "Q" "C" "K" "S" "D" "Y" "L" "*"
## [271] "Y" "S" "V" "Q" "H" "H" "N" "*" "I" "F" "S" "L" "S" "*" "S" "*" "R" "H"
## [289] "D" "C" "L" "K" "I" "L" "R" "C" "L" "L" "Y" "L" "L" "F" "*" "T" "S" "R"
## [307] "I" "C" "I" "L" "Q" "F" "Q" "P" "Q" "*" "D" "*" "Y" "I" "S" "F" "L" "C"
## [325] "L" "G" "H" "I" "P" "W" "V" "I" "*" "L" "R" "F" "S" "D" "L" "I" "Q" "P"
## [343] "F" "C" "A" "W" "N" "L" "S" "H" "V" "L" "L" "L" "S" "L" "V" "P" "I" "A"
## [361] "Q" "V" "S" "Q" "L" "R" "*" "*" "T" "F" "F" "R" "*" "R" "L" "L" "Y" "Q"
## [379] "I" "S" "L" "C" "Y" "F" "*" "I" "*" "H" "Y" "I" "H" "P" "L" "F" "L" "E"
## [397] "W" "N" "V" "S" "L" "I" "L" "N" "E" "*" "I" "D" "L" "I" "Y" "K" "Q" "E"
## [415] "*" "Y" "Q" "I" "V" "F" "S" "S" "Y" "L" "L" "S" "F" "F" "E" "K" "Q" "V"
## [433] "V" "L" "Y" "F" "S" "H" "Y" "I" "L" "H" "H" "S" "N" "H" "L" "Q" "L" "P"
...GC(plasmid_nt[[1]])## [1] 0.3437746plasmid_nt[[1]][1:100]## [1] "g" "t" "t" "t" "g" "g" "t" "c" "a" "a" "t" "t" "a" "a" "a" "c" "a" "c"
## [19] "a" "a" "c" "c" "t" "a" "a" "c" "t" "a" "c" "a" "t" "c" "a" "a" "a" "t"
## [37] "g" "a" "a" "t" "a" "c" "a" "g" "t" "t" "a" "g" "g" "c" "t" "g" "c" "g"
## [55] "a" "t" "t" "a" "t" "t" "t" "t" "a" "t" "t" "t" "t" "g" "a" "a" "a" "t"
## [73] "c" "t" "g" "t" "a" "a" "c" "c" "t" "t" "t" "a" "t" "t" "a" "c" "t" "g"
## [91] "g" "t" "g" "c" "a" "a" "t" "t" "g" "g"comp(plasmid_nt[[1]][1:100])## [1] "c" "a" "a" "a" "c" "c" "a" "g" "t" "t" "a" "a" "t" "t" "t" "g" "t" "g"
## [19] "t" "t" "g" "g" "a" "t" "t" "g" "a" "t" "g" "t" "a" "g" "t" "t" "t" "a"
## [37] "c" "t" "t" "a" "t" "g" "t" "c" "a" "a" "t" "c" "c" "g" "a" "c" "g" "c"
## [55] "t" "a" "a" "t" "a" "a" "a" "a" "t" "a" "a" "a" "a" "c" "t" "t" "t" "a"
## [73] "g" "a" "c" "a" "t" "t" "g" "g" "a" "a" "a" "t" "a" "a" "t" "g" "a" "c"
## [91] "c" "a" "c" "g" "t" "t" "a" "a" "c" "c"rev(comp(plasmid_nt[[1]][1:100]))## [1] "c" "c" "a" "a" "t" "t" "g" "c" "a" "c" "c" "a" "g" "t" "a" "a" "t" "a"
## [19] "a" "a" "g" "g" "t" "t" "a" "c" "a" "g" "a" "t" "t" "t" "c" "a" "a" "a"
## [37] "a" "t" "a" "a" "a" "a" "t" "a" "a" "t" "c" "g" "c" "a" "g" "c" "c" "t"
## [55] "a" "a" "c" "t" "g" "t" "a" "t" "t" "c" "a" "t" "t" "t" "g" "a" "t" "g"
## [73] "t" "a" "g" "t" "t" "a" "g" "g" "t" "t" "g" "t" "g" "t" "t" "t" "a" "a"
## [91] "t" "t" "g" "a" "c" "c" "a" "a" "a" "c"Connect with sequence databases with seqinr
You can query to NCBI, Uniprot and other databases directly using seqinr. The seqinr package was written by the group that created the ACNUC database in Lyon, France (http://pbil.univ-lyon1.fr/databases/acnuc/acnuc.html). The ACNUC database is a database that contains most of the data from the NCBI Sequence Database, as well as data from other sequence databases such as UniProt and Ensembl. the ACNUC database is organised into various different ACNUC (sub)-databases, which contain different parts of the NCBI database, and when you want to search the NCBI database via R, you will need to specify which ACNUC sub-database the NCBI data that you want to query is stored in.
To obtain a full list of the ACNUC sub-databases that you can access
using seqinr, you can use the choosebank()
function from seqinr. Then, you just need to select the
database and query your sequence. However, as you can see in the example
below it does not work currently. I mentioned this here because I used
this previously and found it very useful. In case you want to try it
again in the future you can check the examples in the Reference 4 below.
choosebank()## [1] "genbank" "embl" "emblwgs" "genbankseqinr"
## [5] "swissprot" "ensembl" "hogenom7dna" "hogenom7"
## [9] "hogenom" "hogenomdna" "hovergendna" "hovergen"
## [13] "hogenom5" "hogenom5dna" "hogenom4" "hogenom4dna"
## [17] "homolens" "homolensdna" "hobacnucl" "hobacprot"
## [21] "phever2" "phever2dna" "refseq" "refseq16s"
## [25] "greviews" "bacterial" "archaeal" "protozoan"
## [29] "ensprotists" "ensfungi" "ensmetazoa" "ensplants"
## [33] "ensemblbacteria" "mito" "polymorphix" "emglib"
## [37] "refseqViruses" "ribodb" "taxodb"# select genbank
choosebank("swissprot")
query("pol", "AC=P0DPS1", verbose = TRUE, invisible = FALSE)## I'm checking the arguments...
## ... and everything is OK up to now.
## I'm checking the status of the socket connection...
## ... and everything is OK up to now.
## I'm sending query to server...
## ... answer from server is:
## ... answer from server is empty!
## ... reading again ( 0 ).
## I'm trying to analyse answer from server...
## ... and everything is OK up to now.
## ... and the rank of the resulting list is: 2 .
## ... and there are 1 elements in the list.
## ... and the elements in the list are of type SQ .
## ... and there are only parent sequences in the list.
## I'm trying to get the infos about the elements of the list...
## ... and I have received 1 lines as expected.polseq <- getSequence(pol$req[[1]])## Error in getSequence(pol$req[[1]]): objeto 'pol' no encontradoclosebank()The package rentrez
Like seqinr, other R packages allow accessing to NCBI and other databases, including APE or genbankr. The latter takes advantage of the packages rentrez.
rentrez is an R package that helps users query the
NCBI’s databases to download diverse type of data and metadata,
including sequences or research papers.
install.packages("rentrez")## Installing package into '/Users/modesto/Library/R/x86_64/4.2/library'
## (as 'lib' is unspecified)##
## The downloaded binary packages are in
## /var/folders/6z/hz95tt497dgd98vmjzfch5nw0000gn/T//RtmpEqICx1/downloaded_packageslibrary(rentrez)
(pols <- entrez_search(db = "pubmed", term = "DNA polymerase"))## Entrez search result with 360855 hits (object contains 20 IDs and no web_history object)
## Search term (as translated): "dna directed dna polymerase"[MeSH Terms] OR ("dna ...str(pols)## List of 5
## $ ids : chr [1:20] "36510928" "36510431" "36510378" "36509875" ...
## $ count : int 360855
## $ retmax : int 20
## $ QueryTranslation: chr "\"dna directed dna polymerase\"[MeSH Terms] OR (\"dna directed\"[All Fields] AND \"dna\"[All Fields] AND \"poly"| __truncated__
## $ file :Classes 'XMLInternalDocument', 'XMLAbstractDocument' <externalptr>
## - attr(*, "class")= chr [1:2] "esearch" "list"(Gon <- entrez_search(db = "pubmed", term = "Gonzalez", retmax = 100))## Entrez search result with 61827 hits (object contains 100 IDs and no web_history object)
## Search term (as translated): "Gonzalez"[All Fields]Gon$ids #now kept 100## [1] "36510008" "36509960" "36508433" "36507928" "36507623" "36506217"
## [7] "36505946" "36505806" "36505573" "36503966" "36503720" "36503674"
## [13] "36503356" "36503017" "36502937" "36502882" "36502442" "36502240"
## [19] "36501846" "36501341" "36501276" "36501205" "36500869" "36500780"
## [25] "36500761" "36499474" "36499263" "36499112" "36498916" "36498706"
## [31] "36498045" "36497957" "36497952" "36497826" "36497486" "36497361"
## [37] "36496616" "36496597" "36496391" "36496196" "36496195" "36495858"
## [43] "36495621" "36495405" "36495360" "36494024" "36493726" "36484536"
## [49] "36483758" "36483158" "36483058" "36482892" "36482822" "36482529"
## [55] "36482348" "36482178" "36482170" "36481818" "36481739" "36481724"
## [61] "36481665" "36481658" "36481652" "36481612" "36481610" "36481462"
## [67] "36480764" "36480600" "36480041" "36480023" "36479363" "36479103"
## [73] "36479074" "36479027" "36479009" "36478166" "36476591" "36476528"
## [79] "36476428" "36476293" "36476213" "36475820" "36475411" "36474803"
## [85] "36474107" "36474085" "36473848" "36473599" "36473215" "36472983"
## [91] "36472845" "36472780" "36472393" "36471913" "36471773" "36471457"
## [97] "36471166" "36470964" "36470384" "36469495"# we can also fetch data
her1410 <- entrez_search(db = "nuccore", term = "HER1410")
her1410$ids## [1] "1853362518" "1853362517" "1853362516" "1853362515" "1852678087"
## [6] "1852677931" "1852677628" "1852672161" "183211906"her1410_1 <- entrez_fetch(db = "nuccore", id = her1410$ids[1],
rettype = "fasta")
head(her1410_1)## [1] ">NZ_CP050186.1 Bacillus thuringiensis strain HER1410 plasmid pLUSID3, complete sequence\nGTTTGGTCAATTAAACACAACCTAACTACATCAAATGAATACAGTTAGGCTGCGATTATTTTATTTTGAA\nATCTGTAACCTTTATTACTGGTGCAATTGGATTCTTACCTACACGAGCAGGTTTTGTGACTTCGACTGTA\nATGGAAGATACAATTGCATTTACTGCTGCTTTCTTAGATTCGTTGTTTAAATTGTACCATTCATCTTTCA\nACTGATTTAAAAGTTCTTTTATTTCAAGTGAAGAGGCTGTATCAGTTATATTAGCCAGTTGTTTTTGAAT\nTATGTTCTCTTCTTGGGTTAGGGTTAACATATCCTTTTTATATTCGTTCTTAGGAATATCTCCTTCTATG\nAACAGATATTTTAAACGTGCTTTCTTATCTTGGATTCTGTTATATTGTTCTTGTAAATTACTTAACTCTA\nTTGGTTTCTCTTCAGTATCATCAAGATCAACGATGGCGTCTTCCAAGAGGTTTAGAAATTCTTTTTCAAT\nTACATCCTCTGGAACTTGAGGCATATCGCATGTTCCTTTATGATGTCTTGAGCTACACCTATAGCTCATT\nACAATTCTATTATGGCTTCTTACTTGTTTATTTCCCAAAAAATGCTTTCCGCATCTGGCGCACTTTAAAA\nCATTAGAGAATACGAAAAAATTATGCAATCTAACTTTGCCTCTCTTTCTACTATCTTGTACTTGTTGTAC\nAGTATACCAAGTATCTTTATCAATAAAAGTTTCGAAATCCTTTTGAGCAATGTCTGTTAGAATGTCGTCT\nCCCCAGCGGATTTTTCCAATGTAAATCGGATTATTTATAATATAGCGTACAGCATCATAATTGAATATTT\nTCCCTCTCTTAGTCTTAACGCCACGACTGTTTAAAGATTTTACGATGCTTATTATACCTTTTGTTTTAAA\nCATCTCGAATATGTATTTTACAATTTCAGCCTCAGTAGGATTGATATATAAGTTTCCTTTGTTTAGGTCA\nTATCCCATGGGTGATTTAGCTCCGTTTCTCAGACCTAATTCAGCCTTTTTGTGCATGGAATCTCTCACAC\nGTTCTGCTGTTGTCTCTCGTTCCCATTGCGCAAGTGTCGCAACTAAGGTAATAAACATTCTTCCGGTAGC\nGGTTGTTGTATCAAATATCTCTGTGCTACTTTTAAATTTAACATTATATTCATCCATTATTTTTAGAATG\nGAATGTAAGTCTGATACTGAACGAGTGAATCGATCTAATCTATAAACAAGAATAATATCAAATTGTTTTT\nTCTTCATATCTTTTATCATTTTTTGAAAAGCAGGTCGTTCTGTATTTTTCGCACTATATCCTTCATCACA\nGTAATCATTTACAACTACCCATCCTTGTGATTTAGCATATTGCTCCAGACGTAATTGCTGCATATCTAAT\nGAAATACCTTCTTCAACTTGCATATCAGTAGATACGCGTCTATAAATTACACACTTCATTTGCACATACT\nCCTTTCTTAACTAAATTTCTATATCATATGTTTTATCAAAGCAAATTAGACAAATGTATGGGTCAATCAT\nTTGATTGACTATTTGGCCCAGTGAGAAATTCTCACTGTTCATCCAACTGAATGAAATCATATAAATCTTC\nCATATTTACGTTAAGTTGAGAAGCGATATTCTTAGCGGTTTGATAAGACATGATTCTATCATTATTTGCA\nTAAGAATGAACTTGTTGTTTTTTCATATCTAACTTTAATGCTAAATCTACTTGTGTTAATTTCTTCTCTT\nTTAAAATTTCATTCAGTCGGCATTTGCCGACTACATACACTGTTTCACCGCCTAATTATTGAATGATCGG\nACTCACACTGTATAAAAATTTTCCTATAATTCTTATATCCTCGCAGTTATTTTGTGAGTATTGTTGGTCC\nTTAAAACTTTCGTCATGTGAACAAGGTTCTAAAATTATTAAATCTGTAAATTTATACACCTTTTTTAAAG\nTTGCGTAATGTCCATTTACAATTACAGCTGCTATTTCTCCGTTTTGTACATCACACTGTTTTTTCAGTAT\nGGCGTAATGTCCATTAGGAACAATTTTATTCATGGATTCACCGGTAACAACCAGTCCGAATAATTCATCA\nATACTGTGAGTTTTATATGGAGGTGCAATTCTATCAACTACATCTTCTATAGCTTCCAATGGAACACCAG\nCTGCTATTTTACCAATAATAGGAATTTCTTTTTTTAGTTCACACTCTATAGGTTTTTCTTGAACTAATTG\nAAGTTCCCCATCCACAATTTCAACTTTTGTATTCTTATAAGTTGTATCAATATCAGATTTTGTTACTCCA\nAATACAGCGGCCATTTTTTCTAAAACCCCTGAACTTGGTTTGGCTCTATAATTCATATAATCACTTAAGG\nTACTTCTTGCAATTCCTATTTGACTAGCTAAATCAGATTGAGTCATATTATTTTTTTTTAAAAAATTCTT\nTATGTTTCTTACTATGGTTTGTTTTTGTATATCAGTCATACCGTCACCTCCTATTCTGACTACGCTTTTT\nAATATAACATATTACGAATTATTCGTAAAGCGTTATATCCGTATGGTTTTTTCGTATTTTTGTATTGAAA\nTTACATAAATTTAGTAATACAATAAATCTCGAAGGAAGGGGGAAACGAAATGGATTATTTGAAAAGAACA\nTTGAATGAACTTAGGGAAAGCGCAGGGTTCAATCAAGCAGAGCTTGCGGATATATTAGAAGTATCCCCAA\nAAACGCTATGGTTATACGAACAAGATTCAACAAACATACCAGATGAATTAATTAAAAAGTATATGTATTT\nGTTCGATGTTCCGTATGAGGATATATTTTTTGGTTCTAAGTACGAAAAATTCGTACAAGTGAAGAAACGT\nGTACAAGAAAGAGCAAATAATTTAAAAAAAATTGTTTCGTAAAAATTTGCTTTAAATGGGTAGTCCTATT\nGTCCCACGAACATATAAATGCATTGAGGTGATTGAGGATGAGTGGGGGACAAATCATTCGTGATGAAAAT\nGGGTATGTGGTGAAAGTAATCCTTACAAAGGAACAGTGGAAGAAATTTCTAACACCGTTAATACCAGCTG\nCACGGGAGTTAATAATTCAAAGAAAAGTGGAACAACGAAAAAAAGCAAAATGAATAATTTTTTTAATATA\nTTTGCGAAATTTGACGATAAATATTGGTTCTACAAATTAAAGGAGAGTGAAAAGCATGAATGGAGTACTA\nCTAGCAACACGAATTATGAAAGGGCATGAAGTTGTTAAAAAATGCGCAGAAGCAAGAAATAACCCTTTAC\nTATTAGAAGCTATGGAATCAGAAGCAAAACGTAAGTTATACGAAATGAACTGTAAGGTTTCAAATCGAAG\nACCAACTATGAAAAGTCAAGGGGACATTTTCAAAAAATATTGCTAGAGGAGGTGAGTTAATTGAAGGAAG\nTAACGGTAATTTTTAAATCAGGTGCTAAAGCAAGTTTTACAGTAGAGGAATTTGCGACATTTAAAAATGG\nATTTGGAGCTTTAACAAAAATCGAGTATACAGGTGCTAATAAGAAAGTACCCTTCCACATTGGATTAAGT\nAATATCGATGCAATATTTGTGGAAGACATTGGTGGAAAGGAATCTACTAAAGAACCTGATCATCCAATTG\nAAGATTTCTATGGTTGTGAAATTAAGCAAGATGATAAGTATTTTATGTTCGGACAAAATGCCGTGCTTGA\nAGGGAATCTAACAAATTACTTAATTGCGGAACAAAATGTTGAATGCTTTCGAGCTGTATAAAAAGGAAAG\nGGCGGTAAGGAGTGTTGGCAAATGATTTGTTTGAAGAGAAACAACATCTAGTGTTTGCTGCAATTAAACA\nACAATTTGGAAGTATGACAAGGGCTGCGAAAATCGCAGAGTTAAACAATATGGAACTTGATGATTTAATT\nCAATTCGGTCGTATGTATTTATGGGAGCGTTGTTTAAAGCATGATCCAGAGAGAATAGAAACGTTTAGTG\nCATACGTAATGAGAGGTATGAAATGGGCCATGAGTGATGAACTTCACTTGAAAGGGAGCCTGTTTAAGAT\nTAGCAGACGGATTAGTCATGAAGAAAGGAATAAAATCAACATTCATTCGATTGACTATCATCAAGAGGAG\nGAAGAGGTACACGGATTTTATGCTGTGTCTCCTATCGATGTGGAAGAAGAGGTAACCAAACATATCGAAT\nTTGAAGAAGTAACGAGCGTTCTTGAAGAAAAAGAAAAGTCCATCATTATGCACGTTGGTGAAGGGTATAC\nCACAGAAGAGATTGCTGTGAAATTAGAAATGAAAAAATCAACAGTTCATACAACGAAAACACGTGCGTTT\nAAAAAGATGAACCCAGATTATAAGCCAATAAAACAAAAATCCTTTTTCTTGGGAAAGAGGATAATAAAGA\nGAAACCGCCAGTTGGGGCTGACGGTCTAATAAAAACACATGTTGAGGTCATTATAGCATGAATTGATTTT\nGTGTAAAGGAGAGGCTATGTCTTGAAGAACGGTAAAAAGCCAAACAAACGTGAAAAAATTCATATTCAAT\nCACACGATTTAAATCCACAGGATTGGTTGATTTATAAGAAAGTAAATAAGGAATTGCATTTGGTCCATCG\nGACTACTGGTGTAACTCGTATAATTCCAAATTTATAGATTAGGAGAAAGATATAAATGGCTAATGAAATC\nACGCCTAAATTAGTTGCTGATATAACAGATAAAAGTTTTGGCATACAGCTAATTGTTACTGGTCTTGCTC\nACTTAGTAGAGGAAGAGGGATATACACCACATGAAGCATTAAGTATCGCTCGTTACACAGGAAATAATTG\nTTTCCATGCGTTAGCAGAATTGAAGAAGGAGGCTAAAAAATGAACGACAAACCAATTGATGAATTAGTCG\nCGGAAAAGGTTATGGGATGGATAAAACCACCTGAAACATCAATCCTTAAATCAATGTGGGTTTCTAAGCC\nAATAGGTATGGTTCATCGTGAATTACCTAAATTTAGTATGAATATGAAAGATGCATGGTTAGTTGTTGAT\nAAATTACAAGAGTCATTTAAATCAGTGGAAATATTTATTGAAGACAGAATGACAAATGTGATTATAACTG\nAACATTTCCCAAGTGGACATCTAAAAGATAAATATCAGGGATATGAAAGATCGGCTCCTTTAGCTATTTG\nCAAAGCTGCATTAAAAGCTGTAGGGGAGGAATGGCAGTAATGGATATTAAATCAGTTGCTAAAGCTGTAC\nAAGTTATTCGAGAAGCACAAAATGAACATGGAATCATTAGGATTGATGGAAAGGAAGTACATCTAAAAAA\nTGAAGTATTGGAATCATTGTTAGATGAATCTCAGACTAAGCCTTTAATACTAAGACGTGAATCCAAGTAT\nTATCCTTATGAAGTTTCCTTTATTAATGATCATGTAACCTATTTCTCTCTTTACACTGAAGAAAGAATGA\nAAGAGAAAATTGGAGGGATACCAAATGCTCGAAAATTCAATGGTAATCGGGAACCGTCAGGACTCCCCAT\nTCAATAACGTGATGGATCATTGTCAAAGTTGTGGTAAAGAAATCTATTTTGATGAAGAGTACCGAGATAT\nTGATGGTGATTACATACACGATGAAACAGATTGTATCAAACAATATGTAGAGTCTCATTCCATAAAGAAA\nGTAGCTGGTGAGTAAAATGAACGCTGCTATTGAAGAATTAGAAAAGTCATTATATGTGGAGCAAGGGAGA\nTTAAAAGACTATCACGGCAAGCTAGAGAGGGTAATAGAAAGAAAACCAATCCTTGAAAAGAATATCCAAG\nATACTGAAAGCAAGATCCAGGACATTGAAGCTTCAATTTTTGTTCTGAAAAATATGGTGAAGGAGTGAAT\nTAATTGGAAATCACAAATGGTGCTGAAATTATCAAAAGTGAAAAAGCAAAAATTATCATTTATTCAAAAC\nCAGGTAACGGTAAAACAACGGTTGCTGGATTGTTACCAGGTAAAACATTGGTGTTTGATATCGATGGGAC\nAAGTCAGGTGTTATCGGGTTATAAAAATGTAGATGTAGCTAAAATTGATGGTGAAAATCCACATGATAGC\nATTTTACAATTCTTTGCACTTGCTAAAACAAACATTGGTAAGTATGACAACATCTTTATCGATAACTTAA\nCGCATTACCAAAAGTTATGGCTGCTTAATAAAGGTGAAAAAACAAAAAGCGGTATGCCTGAATTAAAGGA\nCTATGCTTTACTAGATAACCACCTTTTAAAGTTAGTAGAAACATTTAATTCATTAGATGCAAATGTTATT\nTTCACAGCTTGGGAGACAACAAGAAATATCACTCATGATGATGGTCAGCAATATACACAATTCATTCCGG\nATATTCGTGATAAAATCGTAAATCACATTATGGGAATCGTTCATGTTGTTGGTCAATTAGTGAAAAAGGC\nAGATGGTACAAGAGGTTTTGTTTTAGAAGGTAATCAAAGTATTTTTGCTAAGAATCATTTAGATCAGCGT\nAAAGGTTGCGTACAACAAGAATTATTAGTGTCATCCACAAACTAAAATACAGGGGGAAATAAACAATGAG\nTTTTAAATTTAAATTTGATGAAGAAAATGTATCACAAGGGTTTGGATTGGTAGAGGAAGGTAAGTATGAA\nGTTACAATTATTACTGCTGAAGCAAAAGAATGGCAAGGTCAATATTCTATTGGATTTGATGTGGAAATCC\nGTTCAGATATTGAACAAAAACATCAAGGAGCAAAAATCCTATATAACACTCTATATCTAACTACTAACAT\nTCAAGAATATGCAGAGGACACGGAAAGAAGACGTAACTCATTCTTAAAAGCTTGCGGATACACAGGTAAA\nCAGGATTTGGAATTAAATGTTGTTGTACGTGAGATTGTAGGTAAAACGGTTTTAGCTTATGTAAAACATG\nAAACAAATAAGGATGAAAAAACATTTGCTAAAGTTAAATTTGTGGCACCATCGAATGTAAAGCCGTCTGA\nACCAAGTGGACCACCGATTACTGTTGGCGATGATGATTTACCATTCTAAATAACTAAATAGAGAGGTCGG\nTTTTGTCGACTTCTCTTTTTTATACCCTAAAAAGTTAATTGGAGGGCGCAATGAAAGAAAATCCATACAA\nTTTTAATGAAATTCCTGCTGAATTAAAGGGCCTTCCCCAGTGGATCTTATGGAAGCTTGAAACCAGAAAT\nAATAAACAAACAAAAGTACCCTATCAAGTAGATGGAGAAATGGCCCAAGCAAATAATAGACGTACCTGGT\nCAACATTTGCAACGGCAGTCAAATTTTATTTAGAAGGTGACTATGATGGAATCGGGTTCGTGTTTAGCAG\nGCAGGACAATTACATCGGAATCGATATTGATAAGTGTGTTACGGACGGAAAAACAAATGCTTTTGCAACG\nGAAATTATCGATACATTAGATAGTTATACGGAGTTTTCACCTTCAGGAAAAGGTATCCACATCATTATCA\nAAGGGAACCTTCCACAATCTGTTTTAGGTACAGGACGAAAAAATACAAAGCACGGTTTAGAAATTTACTC\nATACGGACGTTTCTTTACCTTTACTGGAAATCGTGAGAATTCCAATGATGTATACGAGCGAACGGATGAA\nCTAGCTGAAGTATTTGAAAAATATTTTGATGATAGCGACATTCAAGGTCGTGTAAATTTAGCAGAGTTTG\nAAAAAGATGAAATCAAAATTTCGAACGATGCTCTATGGGAAAGGATGTTTAGAAGTAAGAATGGTGATGA\nAATTCGCTCGTTATACAATGGCAGCTTAATAAATGATGATCATTCAGCAAGTGACCTTTCTCTATGTAAT\nCATTTAGCATTTTGGACAGGGAAATCAGCAATTCGAATGGACTCCATGTTCCGTGAGTCAAGCCTTATAC\nGTGATAAATGGGACGTTATTCATTTTAGCGATACAAATGAAACATACGGTGAAAGAACGATAGCAACAGC\nCATTTCATCTACTTCCACAACTATTTTAGATAACAAACAGCAATTCGAAGAATTTTCTTTTGATTTCATG\nAATGAAGATGCGGTTGAAGTTGTGGAGGACAAACCGAAAAAGAAATTCCGTTTAACTGAATTAGGAAACG\nCTGAACGTATCGCATATGAATATGGCCATGTAATCAAATATGTTAGCGATATTGGCTGGTACATATGGGA\nCGGTAAACGCTGGAAGTTGGACACGAAAAAAGAGATTGAAAGAATTACAGCAAAAGTACTTAGAAGTCTT\nTATAAATCAGAAGATGAATTAGAAACAAAATGGGCTCGAATGTGTGAACGGAGAAATATTCGTATGAATA\nGCATTAAGGATCTTATGCCATTGGTTCCAGGTGAGCGTGAGGACTTTGATAAGTATAAATACTTGTTCAA\nTGTTGAAAATGGCATTGTTGATTTAAAAACAGGAAAGTTGCAGCAACATGATCGGGAACTTGGTTTAACT\nAAAATTACTAATATTTCATTTGATGAAAATGCAAAATGTCCAGAGTGGCTTAATTTCTTAGATCAAATTT\nTCCAAGGTGATAAGGAACTAACTGAATACATGCAGCGGTTAATTGGTTACTCACTAACAGGGGAAATCAC\nGGAGCAAATAATGGTCTTCTTAATTGGTGGAGGTTCCAACGGAAAATCGACCTTTATTAATACCATTAAG\nGACCTTTTGGGTGAATACGGTAAACAAGCAAAATCAGATACTTTCATCAAAAAGAAAGAAACTGGTGCCA\nATAATGATATTGCTAGATTAGTAGGGGCACGCTTTGTATCTGCAATCGAAAGTGAAGAGGGTGAACAACT\nCTCAGAAGCTTTTGTAAAACAAATAACAGGCGGTGAGCCAGTATTAGCACGTTTCCTTAGACAAGAGTAT\nTTTGAATTCATACCAGAGTTTAAAGTGTTCTTTACAACAAACCATAAACCAGTAATTAAAGGTGTAGATG\nAAGGTATTTGGAGACGTATCCGTTTAGTTCCATTTAACCTGCAGCTACCAAAAGAGAAACGTGATAAGAA\nATTACCAGAGAAAATTAGCTTAGAAATGCCAGGAATCCTGAATTGGGCAATTGAGGGTTGCTTGAAGTGG\nCGGAAGTCGGGACTAAACGATCCAGCAATTGTTATGAAAGCAACAGGTGATTATAAAGAGGAAATGGATA\nTTCTTGGTCCGTTCATGTTTGAATGTTGTTTTAAAAGAGAGGATGTCCAAATTGAAGCAAAAGAATTATA\nTGAAGTTTATGCGAATTGGTGTTTTAGAAATGGTGAACATCAATTAAAAAATAGAGCCTTTTACCGAATT\nTTAGAATCCCAAGGATTCAAAAGAGAACGTGGCAGCAAAAACAAGTATTACATCAAAGGTGTTACTCTAA\nCCGACCGAAAAAATACTTTTAAGCAGCAAAAGTTACTGAATTTCGATGAAAATAGCAAAAGTGTTACTAA\nAAGTAACCCATTTAAAATCACTTAAAACCCTTGATACATAAGGGCTCAAGATACTTTTTATATTCCTTTT\nGTTACTTTTGTTACTAAAAAATACTATAAACAAAAAATAAATATATATATAAGTATTCTATTAGGAGCTT\nCTTTGAACTTTTTAGGTAACCTTAGTAACCCGAAAGTAGCATGAGCCGTTGGGGCTGTAAGGCTCAAGGC\nGTGTTACTAAATGAAAAATAAGAGTAATTTATGGTGTTTTTTAAGTAAAAAACGGTTATTTAAGTAACAC\nACTTAGTAACGGAGGTTGTATTAAATTGGAAAAACATTATAAAGAACACGGGCTAGAAGATTTTTGGAAA\nTTTAAAATTATACGATCCAAGAAAAATGAAATTGGTTGTTCAGAAACCTATGAAAAATATAAAAAATGGT\nGCTATGAAAACAATACTATTCCGAATAAAAGAATTACATTTTTAAGATTCTTAGAAACAAAAGGGTTTGA\nAATTGTGAAGAGCAAAAAAGAACCATTGTATTTTAAAGGAATGAAGATTAAATGGTGAAAGTTTTATTAA\nTTTTAAGTGCGATTTGGAAATCAGGTGCAGATATCTATCTTGATAAAAAGGATAATCAAGTTGCGATAAA\nAAAACAAAATTTAATTCCAGCGGAAGTAATGAAAGCCGCTGAACAAAACTATCAAGCTATTTATGATTGG\nTTTCAATCTTGGAAAGATGAGAGTGCGGAGAAAATTACGTTAATGAAGATATTTCATCATTTTTGTGGGT\nGGAAACATAATCAAAAATTACACAATTGGTTAGTTGAAGAAGAAGATTCATTGCAACTGTTTTATGAGTG\nGACGATTGTCCTTGCTAATAATGGTTGGACAGATGTTTATGAGGATCATCGTCAGTTTGAAAATGATGAA\nTCAAATGCAATGGCAAGAAAGATATATGAACGTGCGGTTTTATATGCAAGGAAAGGGGCATGAAAATGAG\nAGAGATAAAATTCCGTGCTTGGGATGGAACGAGTTGGGTTTATAGCGAATGTATATCAAAAGATGGTATT\nAATTGGTGGATATTAAATAATGAAGATGATAATTGGTTGACGTGTTTAGATCCACAGCAATATACAGGTT\nTAAAAGACAAGAACGGTAAGGAGATTTATGAAGGAGATATTACAAAAGACAAGTTTGGTAATCTTGATGT\nTATTTGTTGGATTAATAGTTCAGGTGCATATGCGACTGTGTCGATTAATTTGTATTTAGACGGAGAGTAT\nGAACATACGGTTGTTGATGAATTTGGAACGGATTGTTTCTTTGAGAATAATGTTCCTGGTGATTTTTTAG\nAAGTAATCGGAAATATCTACGAAAACCCAGGGTTACTGGAGGATTCAAAATGATTCGTTTCCATTACACA\nGATAAAGAAATAGAAAAAATCCTTAAAACACTCACTATCGTTATCGATACTCGTGAAAATGTAAATGACC\nACATTCGTGATTACTTATATCAAAAGGGCATCCCAATTAAAAATCAAAAATTAGATACCGGTGATTATGG\nTTGCATGATTCCCAAAAATGAAGAGTTAGGAATACCACGTGATATTTATTTAGATCGTCGGATAGAAAGA\nAAAATGAGTATCGATGAAATTACAAGTAACCTACAAAAAGATACGCAAACAAGATTTGAAAATGAATTGA\nTTCGATCGAAGGATATTCCATTCACTTTAATTGTGGAAGATCAACGTGGTTACGAGAAAATACTTAAAGG\nTGATTATAAATCAAGATATAACCCATTGGCATTACTCGGTAGGCTTAATACTTTTAAAGCAAAATATGAT\nTTTGAAATAGTCTATCTAGATAAAAAGTTTGTTGGTAATTGGATATATTATGTCCTTTATTATCATGCAA\nAACATTATCTTAAAACAGGTGCATTTTAAGTTCAGAAACGTACAGAAATAACAGAAAAACTTTAGTTCTA\nATATCAATTTAATATAAAAGGAGTCGGAAGACATGACAAAGGTAAAATTGAACGTATTATTCAAGAAAAT\nGCAAAAGGATGATAAAAAGGAAGTTTTGATGTTCCACGTATTAAGTGATGAATTACCACATGCTGATGAG\nTTATTGAAGATGCCAGGTACTATTGTTTATCTAACTGTGGAAAAAAGCGATGTTGAAGCAATTGGTGCTG\nAATTTGTTTCTATTCAACGTGATAGTAAGAAAACCGTTCTTAAATTCAATGTAAAAGGCGATACGAAAGA\nTAAAATTAATAAACTTTATCCATTCGCTGGTGAAAATGTTTCTATTACTCTAGAGCCTTCGCAAATGTCG\nATTGATGAGTTTTACGAAGAACAACATGAAGGAGTGGAGTATAACGTTAATCCTGATGGAACAACTGATG\nTTGCTCCGGGGCAATTGAAGATTGTTGATGAAGAAACGATTGCTGAATAAAAATTTATCCTGGGCTTCGG\nCTCAGGATATTAATACATTTTGAATTTTGTTAAGAAATGAGGGTAACTTTTGAAAATTATAAAAGATGAT\nAAGAAGTGCCAAACATGTATTTTCTATGGTTTATGCACGAAAGCGCACTTACCACACTGCCTAGGAAATG\nATTACTTCAAAGACAGTGTCCAAAAAGAAAAATAAAACTAAAGCTAAAGCGTTACTTTAATCGGAAATGG\nCAGGTAATTGACCAAATCACCTGCCATTTGCCTAAACAGTCCGGAGGGGTAAAGCTCCGTTTTGAAAGAG\nTGTAGCCGACTCGTAGATAGTATGTGTAATGTAAAAAAGATTATTCGTATATCACACGGGATATGAAAAG\nGAGAATGAAAAATGTCTAACAAATTTGATAAGGACTTTGAAGCAATTACGAATAATGGTGAATTAACGAA\nAGAAGGAACAGAATTTATAGAAGCTGTAAAGCAATTCGTCGGTACGCAATATGATTCAGAGGATTTAGCG\nAAATTAATGGTATTAATTATGGCTGCTTTAGATGTGGATGACAATGCTTTTAATACAGCGATATCCGCTT\nTATATCAAACTGCAATAGAAGTGCAAACAGGTATCAATCTGAATGAATTACTGGATTTAGTGACAAAAGG\nTGAACCAGGCTTAACTCATTAGGAGAACCCAATGTATTCGACGAGTTTCGACATGAAAACGACCGTCAGA\nAACATAGTGATTTGAAGTTTTATTTCTTTCTGAATACAAATAGGTGCACAAGTGTTAAAACGTCTTAGAA\nAGGAAAATAAACGTGTTTTATGAGATGTATTGTTTTTTATAGAAAGTAGGTGAATCATCATTTGTTTGAC\nTGGCTAAAAGACTATCAGAAATTAGAAGAAGACATTGAGTACTTAGATTACAACTTAGATAAAACAAAAG\nCAGAATTAAAACGCTGGGTCAGTGGTGATTTGCGAGAGGTACGTTTAACTGCTGAATCGGAAGGTGCAAA\nGGTAGAAGAACGTATAGAAGCAATTGAATATGAGTTAGCGCACAAGATGAATGCAATGCAGGATGTATTG\nAAATTGATAAATAGGTTCAAAGGTTTAGAAAATAAAATGTTAAAAATGAAATATGTGGACGGAATGACAT\nTAGAAGAAATAGCTGAGAATATGAATTATAGTTCTAGTTATATCTATAAGAAACATGCTGAAATAATAAG\nGAGAATAAAGTTTGCTGAAGAACTTGCACTTTACTGACACCCAGTTTTATGAATGTTAACTCTTGAAGAT\nATCGATTATAGTAATAACATAAGAAATTGACGAAAGGGCAACTGGTGCACAGTTGCTCTTTTTAATTAAA\nCAAACCATATCCATATAATAATGATAACAGCAAAGATAATGATTAGTATTTGTGAGAATTTCATAACATG\nCTCCTTTTAAACTATAGTGTTTGCAAAGGATTGAAGAGGCATTCCTTATGGAGTGTTTTTTATTATGTAA\nAAATTATATAGGTGGTGTAGAGATGACTTTAACGTTGCATAATGGAGATTTGAATAAGTTAGCAAGAGAT\nACTTCACAGGACAGTATCATCTTGAGAGTTGGTGAACAAGAAATGGTATCTCTGAAAAGCAATGGAGATA\nTCTATGTTAAAGGTAAGCTTGTTGAAAACGATAAAGAAGTTGTAGATGGCATGAGAGAGTTATTGATGTT\nATCTAGGAAAAGGTAAGCGCAAACGTGTTGCATTTTATAAGGATGGTGAAAAGAAGATGTTCTCATATAA\nAAAACGTATTGAATCCTTAGATGAACTTCATAAAGTTATTGATGCATTAGAAACGTTAAAGAAGGAGTAT\nCAGATTATCAAAAAGATAGAATGGTCGGAACCTAAATCACCTCTTCCTAAATTACCAAAGAATGTTTGGT\nATGTTGAAGAAGTTGATACATCTAAAATCTTTTTTACTGATGCTGATGCAATAGTTAAATTAGTTTGTAA\nAAATTGTGGAATATCGTGTAGAGGGAAAAGAAATCTATTAGAAGGAATGAGTTGTATTTATTGCTTTAAT\nCACAATACAACAATAGTTTCTTTAGATAAGGAGTTTGAATGTAAATGAAAGTAATTATGGGTGAAGAACC\nTTTATTACCTAATGGAAAAAGAAATGGAGAAGCTGTTAGAAGTATGCTTGATAAAATTAGGTCGCAAGAA\nCAGAAAGATAAGGAGAGTGAATGCAAATGATTACTGAAATTAGAAAAACAATATCTGGTACAGAGTATTG\nGGATAACAAAGAAAAACGAAGTCTATTTGTTCCTACAGATGAAGAACCAGGATTCGAAGTAACTGTTAAT\nCCTGAGAGTATGATCTTAGGCATGGACATATCAAGTGAACCTGATAAGACAGTAGTTAATTTAAATGGTA\nTGACAGTGAAACAATTACATGAATATGCTGCATCGATTAATGTTGAGATTCCAGCCGATGTTAAAAAGAA\nAGAAGACATCATTGATTTACTATCATGAAGTACTGTGCTGAACAAGGCTGCAAGACATTAATCGATAAAG\nGACGATACTGTCTCAATCATAAACGTAAACAGAAGAAGACAGTTGTGTATTCAAAGAACAGATCATTCTA\nTCGTACAAAAGCCTGGCAAGATTTAAAGTCATTCTGTTATCAAAGAGACAAAGGATTGTGTCAACGATGT\nGGAAGGTTTGTGTTTGGTAAGCAAGCACATCATCATCATATTGTTCCAATTAAAATCAATCCTTCATTAA\nGATTAGATCCAGATAATATCGATACACTTTGTTCTAAGTGTCATCCGATTGTAGAAAGAGAAACAAATGA\nAAAATACCAGGAAAAGAAAAAGTTCGACTGGAAACTATAAGCCCCCCCTATCAAAAAAAGAAAAGCTGGC\nCTTATGGGGGGATAGGGAGTGGGGGTGCAAACGCGCACCTCAAAATGGTTTTTTGAAAAAAATTTGTTTT\nTTTTAGGTGGTGATTTAAGGAATGGCCAGAAAATCGAAGGTCGTAATTGAAGCTGAAAAGAAAAAAGAAT\nTAGAAGCGCAGCGTATTATGGATGTTTTGGTTGAAGCCGGAACTTATTCGCCAGCGCTCGATCCATTAAT\nTGAAGTTTATCTTGATGCAGTTGAGATATACAGCGTCAAATATGGATTGTGGAAGAATTCTAATTTTCCA\nACAGTCCAAAAAACAAAGAATGTAAATGGTGATGTGAAAGAATCAAAGCATCCATTAGCTCAACAAGTTG\nAAGTTTGGTCTAAGCAAAAAGCGAAATATTTGGGGCAATTAGGACTGGACGGAAAGAACAAAGATTTACT\nTAAAAAAAGTGGGGTTCTTCTCGAAAAAGGAAAAAATGAAAAAGAGCCCACGGAGTCTACTGATAACAAC\nAAATTATTGCAATTTAGGCAGAGGTTAAATAGATGATTGATTTTGAAACAAATTACGCTGATATATTCGT\nTTCGGAAGTAGATGCAGCCCCGCACTTATATCCTGATTCAATTAAATTAGCAATCAAACGATATAAGAAA\nTGGAAGAAACGAAAAGATATTTGGTTTGATGTTGAAAAAGCAAATGCTATGATTTATTTCACAGAAACAT\nTCTTAAAACATGCAAAAGGAAAATGGGCAGGACAGCCGTTAATTTTAGAATCCTGGCAAAAGTTCTACTT\nTGCTAACATTTATGGATGGCAAAAATATAATGAAGATGGTAAAGCGGTGCGAGTGATTCGTACGGCTTAT\nTTGCAGGTTCCAAAGAAAAACGGAAAAACAATCATGGGCGGTTCACCTGTTATTTATGCGATGTACGGTG\nAAGGTGTAAAAGGCGCAGATTGTTATATTTCCGCTAATACTTTTGAACAATGTCAAAATGCAGCTGGACC\nAATTGCATTAACTATTGAAAATAGTCCAGATTTACGGCCAGATACACGTATCTATAAAGGCAAAGAAGAC\nACGATTAAATCAATTAAATACACATTTGTGGAGGATGATATAAAATATGCAAATGTAATTAAGGTTTTAA\nCGAAAGATAACGCTGGTAATGAAGGTAAAAACCCGTATATTAATTATTTTGATGAAGTTCATGCTCAAAT\nGGACCGTGAACAATATGATAACTTACGTTCAGCACAAATTGCCCAAGAAGAACCACTCAACATCATCACT\nTCCACAGCAGGGAAGAATACCGGCTCGCTTGGAACTCAAATTTATACCTATGCAAAAGAAGTTTTGGATA\nAGGATAAAGATGATTCTTGGTTCATGATGATCTATGAGCCGAATAAAAAGTTTGATTGGGAAGACCGTGA\nTGTTTGGCGAATGGTTAACCCGAACATGGATGTATCAGTTAACATGGAGTTTCTTGAAAATGCATTTAAA\nGAAGCTCAAAACAACAGCTTTAATAAGGCGGAGTTCTTATCAAAGCATTTAGATGTATTTGTTAACTATG\nCTGAAACATATTTTGATAAAGACCAATTGGATAAAATGCTTGTTGATGATTTAGGAGATATTGAAGGTTT\nAACTTGTGTTATCGGTGTGGATTTATCAAGACGTACCGATTTAACTTGCGTATCGTTAAATATTCCAACA\nTACGATGAGGAAGGTCTTTCATTGTTAAAAGTAAAACAAATGTATTTTATTCCAGAGTTTGGAATTGAAG\nATAAAGAACAGCAAAGAAATGTTCCATATCGGGAATTAGCTGAAAAAGAATTTGTGACGATTTGTCCTGG\nTAAAACGGTTGACGAAGAAATGGTCAATCAGTACGTTGAATGGGTATTTGAGAACTTTGATTTACGTCAA\nATTAATTATGATCCAGCGCTTGCTGAAAAGCTTGTTGAGAAGTGGGAAATGCTCGGTATTCAATGTGTGG\nAAGTTCCACAGTATCCAACTCATATGAATGAACCATTTGATGATTTTGAAATCTTATTGCTTCAGGACCG\nAATTAAAACTGATAATCAATTATTAATTTATTGTGCAAGCAATGCAAAAGTAATAACTAATATTAATAAT\nTTAAAAACACCATCTAAACGAAAATCACCGGAGCATATCGATGGGTTTGTGGCCATGTTAATTGGTCATA\nAAGAAACGTTGAATATGATGGAAGATGCTGTTCCAGATGAAAATTATGATGAATATTTAGATGATATTTA\nTCGATAGAAAGGCGGTGAGGAATTGGGTTTAAAGGATAGATTTTCAAGTTTTTTACTTAAACAAGCTGAA\nAAGCGTGGTTTATTCGAAGACATTTTCAATAATGTTGTTCGCTATGGTGGTAGATATGCAGGCGATGATA\nATATCTTGGAATCTAGTGATGTTTATGAATTACTACAAGATATAAGTAATCAAATGATGTTGGCTGAGAT\nTGTTGTGGAAGAAAAAGACGGTAAGGAAATTAAAAATGATTCAGCTCTTAAGGTTTTAAAGAATCCAAAC\nAATTATCTTACACAGTCTGAATTCATTAAGTTAATGACTAATACCTATTTACTTCAAGGTGAAGTCTTTC\nCAGTGTTGGATGGTGACCAATTACATTTAGCATCTAATGTTTATACAGAATTGGATGATAGATTGATAGA\nACATTTCAAAGTGAATGGAGAAGAAATTTCATCATTTATGATTCGACATGTGAAGAATATTGGTGCCGAT\nCATCTAAAAGGTACAGGCATTCTTGATTTAGGTAAGGATACACTTGAAGGTGTTATGTCAGCTGAGAAAA\nCTTTAACTGATAAGTATAAAAAAGGTGGTTTATTAGCATTCTTACTTAAGCTAGATGCTCATATTAATCC\nACAAAATGGAGCGCAGTCCAAATTAATTAAAAAGATTTTAGATCAATTGGAATCCATCGATGATGCAAGG\nTCAGTTAAAATGATTCCACTCGGAAAAGGATATTCAATAGAGACGCTTAAAAGCCCGTTAGACGATGAAA\nAGACCCTGGCCTATCTAAACGTATACAAAAAGGATTTAGGTAAGTTTTTAGGCGTAAATGTGGACACATA\nTACGGCCTTGATTAAGGAAGACCTTGAGCAAGCAATGATGTATTTGCATAACAAGGCAGTTAGACCGATA\nATGAAAAACTTTGAAGACCATTTGAGTCTTCTTTTTTTCGGAAAAAATTCGGATAAACGTATTAAATTCA\nAGATAAATATCCTTGATTTTGTTACTTATAGCATGAAAACAAACATTGCTTACAACATTGTTCGAACTGG\nTATTACTTCACCAGATAATGTTGCGGATATGCTTGGATTCCCTATGCAAAATACACCTGAGTCACAAGCG\nATTTATATTTCAAACGACTTATCAAAAATTGGTGAGAAACAAGCTACAGATGATTCACTGAAGGGAGGTG\nATGGAAATGGCAAAGACAAAGGAAACACGGACATTTGACATCACCAAATTAAGTACCAGGGATGCTACGG\nAAGAACAACCTTCCAAGATAACGGGTTATGCAGCCGTATTTAATTCAAAGACAACTATTGGTGGCTGGTT\nTGATGAAGTTATTGAACCTGGTGCATTTGCTCGTTCTCTTTCTGAGAATGGTGATATTAGAGCGTTATTC\nAATCACAATTGGGATAATGTCCTAGGGAGAACAAAAAGCGGTACATTGAGACTAGAAGAGGATGAAAAAG\nGACTTAAATTCGAAATTGAATTACCAAATACATCTGTTGGTCGAGATTTAGCTGAAAGTATGTCCAGGGG\nAGATATTAACCAATGCTCATTTGGATTTTGGATAACAGAAGAGAATTGGGATTACAGTGTTGAACCAGCA\nTTAAGGACCATTAAAGAAGTAGAACTTTATGAAATATCGGTTGTTTCAATACCAGCTTATGACGATACGG\nAAGTATCTTTAGTTCGCAGTAAAGAGATTGGTAAAGAAATAGAACAACGAATGAAAATGATTAAACAAAT\nAAATCAAATCTTGGGGGAAAAGTAAAATGAACAAACAATTATTATTAGCATTACAAAAACGAAGCAATGA\nAAGATTAGTGGAATTACGTACACAGGTTGAGAATCCTGAATTACGTGCTGAAGACTTACCAGCAATTCAA\nGAAGAAATCGATGAAATTAACAAGCAATTGCAAGAAGTTGCGGATGCTTTAGCAAATCTTGAAGATGATG\nGTGGAGGTGAAGAAGGAAACGAAGATAATGAAGAAGGTGATGAGGGATCTGGTACTGAAGGTTCTGGAGA\nAGGCGGAGAAGGGCGTGCTAGCAATACTGAAGGTGGAGAAAATAGAAATGGATTAACGCCTGAACAAAGA\nAGTGCAGCAATGGCAGCTATTGCAACAGGTCTTTCTACTCGAGGCCATAAATCTACTAAAAAGAAAGAGA\nAAGAAATTCGTTCGGCATTTGCTAACTTTGTAGTTGGGAAAATTACAGAAGCTGAAGCGCGTTCTCTTGG\nTATTGAAGCTGGTAATGGTTCAGTAACTGTGCCAGAAGTTATTGCGAGCGAAATCATTACTTATGCTCAA\nGAAGAGAACTTACTACGTAAATATGGTTCAGTTCATAAAACAGCAGGTGATATGAAATATCCTGTTCTTG\nTTAAGAAAGCAGATGCAAATGTACGTAAGAAGGAACGTAAAGATAGTGATGAAATCGTAGCAACAGATAT\nTGAATTTGATGAAGTGTTACTTGATCCAGCTGAGTTTGATGCACTTGCTACTGTTACTAAAAAGTTATTA\nAAAATGACAGGTGCTCCAATTGAGCAAATCGTTGTGGACGAGCTGAAAAAGGCTTATGTTCGTAAAGAAA\nTTAACTACATGTTCAATGGCGATGATGTAGGTAATGAAAACCCAGGTGCGTTAGCAAAAAAAGCGGTAGC\nGTTTAAGCCTTCTAATCCAGTTGATTTAAAAGCAAAAGATGCAGGGCAATTAATGTATGATGCATTAGTT\nGAAATGAAAAATACACCTGTTACAGAAGTAATGAAAAAAGGACGTTGGATTATTAACCGTGCAGCATTAA\nCAGCAATTGAAAAAATGAAAACAACTGATGGATTCCCATTACTACGTCCAATGACACAAGTAGAAGGTGG\nTATTGGAAATACGCTTATTGGCTATCCTGTGGACTTTACTGATGCGGCAGACGTAAAAGGAAAACCAGAC\nGTTCCAGTTTTATATTTTGGTGATATTTCAGCGTTCCATATTCAAGATGTAATTGGCGCTATGGAATTAC\nAAAAATTGATTGAAAAATTTGCTGGTACAAACAAAGTTGGATTCCAAATTTACAACTTATTAGATGGTCA\nATTAGTTTATTCTCCATTTGAGCCAGCTGTGTATCGTTTTGAAGTGCAAGCTACAACTGAAGGTAAATAG\nGTTATGAATGATTTAATTGAGAAATTAAAATCTCATATTCATTGGGAAGAGGGTATGGATGAAACCATGC\nTCTCTTTTTATATCACTCAAGCAAGGACTTATGTAAAGAATGCGACAGGCAAACAGACCGAATATCTAAT\nTATTATGGTCGCCGGCATTTTCTATGATTACAGGGTCGCTGAAAAAGAATTAGAACAAGCTCTTGATGCC\nTTAACACCAATGTTTGTCCAGGAGGTTTATGCTGATGAAGAGAAAGACGAATAAACTCAAATGGATGGGT\nGAGCTACTTAAATTAGGAGAAACAATTGATCCGGAAAATGACCGAGTTGTTATGGGATATCCATTAGAAC\nGGAAAATTCGTTATAACAACATTGGAGTTACGGCTACTGATAAATTTACAACGAAAGATACGAATGAAAT\nTGTAAAGAAAATTGAAGTTCGTATTGATCGAGATATTGAAAACGATCAAAAGGATTATCGTGTAAAAGTT\nGGTGGCCGTATTTATAACATTGAGCGCATTTATGTGAAAGAAGAAGATCGATTGATGGAGGTGTCACTAT\nCGTATGCAAATTAGTTTTGAACAGTTGCGAAGCCTTATGAAGAAATCTGGTATTCCAGTTTCTCGTGATA\nGTGCTCCTACAGGGATTGATTACCCTTATATTGTGTATGAATTTGTGAATGAGCAACAGAAAAGAGCTTC\nTAATAAGGTTCTAAAAGATATGCCACTTTATCAAATTGCTGTTATTACAAATGGAACTGAAAAAGATTAT\nGAGCCGTTAAAGGCTGTTTTTAACGAAGCAGGCGTGTCTTATTCTCAATTTGATGGAATGGGTTATGACG\nAGAATGATGACACTATCACGCAGTTTATAACGTATGTGAGGTGTATCCAGTAATGGCTTCAAATAACAAT\nGGTTTTGCTGAAGCTTTAGAAGATATTAATACGCTATTACGATTGAATAAAAAGGTCGAACTGGATGTAT\nTGGACGAAGCAGCGAAGTATTTTGCGAGTAAATTAAAACCAAAAATTAAAGCATCCAGTAAAAACAAGCG\nGACACATTTAAGGGATAGCCTAAAGATTGTTGTGAAAGATGATCGTGTATCTGTGGAATTTAAAGATGAA\nGCTTGGTATTGGTACTTAGTTGAACATGGCCATAAAAAAGCAAATGGTAAGGGACGTGTGAAAGGAAAAC\nACTTTGTTCAAAATACCTTTGATGCAGAAGGTGACAAAATCGCTGATATTATGGCACAAAAAATAATTGA\nTAGAATGTGAGGATGATATACATGACAGTTGAAAATAAAGAAATTCAATATTCTGTAGGGATCGAAGATT\nTATATCTGTGCTTGATGAAGGGAAATGAAACTTCTAGTGCACTACCAACTTATGAGGATATCGTTTATAG\nACAAACGAATATTTCTGATTTAACGATTTCCACTACTTCTACTAATTTTACAAAGTGGGCATCTAACAAA\nAAAATTATTAACATTGTCAAAAATACAGCGTTTGGATTAGCTTTTAATCTTGCTGGTCTAAATCGTGAAG\nTAAAAGATAAAATCTTTGCTAAAACACGTAAAAAAGGTGTGTCTTTTGAAACAGCGAAGGCGAAGGCGTA\nTCCAAAGTTCGCAGTAGGGGTTGTATTCCCTTTAAATGATGGAACAAAAATATTACGTTGGTACCCAAAA\nTGTACAGTTGCTCCAGTAGAGGAATCTTGGAAAACACAAGGTGATGAAATGACTGTGGATGACATTGCTT\nACACAATTACAGCAGATCCATTGTTATTTAATGATGTAACACAAGCTGAATTGGATACTGGTGATCCAGA\nGGCAAAAGGAATTAAAGCTGAAGATTTCCTAAAACAAGTCATTTGTGATGAATCTCAACTAGCGCAGCTA\nGGTGGAACGACTCAAACAGGTAAATAAGGAGGGTGACTATGGCACGTTTAAGTGATTTAGTTAACGTTAA\nTATAACTAGGAATAGCATTAAGATACAGGGTGTCTCAATCCCTGTTATTTTCACTTTTGAATCTTTTCCT\nTATGTGGAAGAAGCATTTGGAACACCTTATCATGAATTTGAAAAAGAAATGAATGATATGTTAAGGAAAG\nGTCAATTTAGCCTGGGAGAAAATGAAGCGAAATTGATGCGTGCATTAATTTATGCGATGGTACGTAGTGG\nTGGTACGGAATGTACATTAGATGAATTGAAAGGTGCTATTCCTATGAATGAATTACCTGATATTTTCATC\nGTTGTATACGAAATTTTCAGTGGCCAAACTTTCCAGAATTCTGATATGGAGAAGTTGAAGCAAGAAAAAA\nAGTAAAAAACATACTGACTAAAAACGAGGAATCTCAGTCCGAATTGGACTGGGATTTTTATTTTTATGTT\nGGTAATACGTTGCTTGGTTTAAGTATGAATGACTTCTGGAAAATCACACCTGCACATTTTTTAAAGCAAT\nTCATCATGCATCTCAGATACAACAATCCGGATGCGTTACATGAGCAGAAAACGAAACAAATTTACACGCT\nAGATCAAACACCATTCCTATAAGAAATGAGGTGAGAAAATGCCTGGGAATAGTAAAGAAAGAAACGTTGT\nTCTTAATTTTAAAATGGATGGCCAAGTTCAGTATGCAAATACATTGAAACAAATCAATATGGTTATGAAT\nAATGCAGCGAAAGAATATAAAAATCATATTGCAGCAATGGGCCAAGATGCGACGATGACTGATAAACTTC\nTTGCTGAAAAGAAGAAGCTTGAAATTCAAATGGAAGCAGCTAAAAAGCGTACAGCTATGTTGCGTTCTGA\nATATCAAGCGATGTCCAAGGACACAAGTACAACCGCTGAACAACTCAATAAAATGTATGGAAAGTTACTT\nGATGCAGAACGTGCTGAAACTACTCTTAATAATGCAATGAAAAGAGTGAATGAAGGTCTTTCAGAGCAAG\nCCATTGAAGCACGAGAAGCACGTGGAGACATGGAGAAACTTGAAGCTAATACTAAACAACTAGAAGCTGA\nACAAAAGCGACTAACAAGCTCGTTTAAGCTTCAAAATGCCGAACTAGGAGCGAATGCTAGTGAAGCTGAT\nAAGTTGGAATTAGCGCAAAAACAATTACGTCAGCAAATGGAAATGACAGATAGAGTCGTCCACAATTTAG\nAACAACAGTTAAGTGCAGCAAAGCGTGTATATGGTGAGAATTCCACAGAAGTACAGCAACTTGAGACGAA\nGCTAAACCAAGCTAAAACCACGTTGAAGCAGTTTGAGAATTCATTACAGAGTGTTGGTCGAAGTGGAGAT\nCAAGCAGCAGATGGTATGGAGAAATTAGGAAAGAAGTTAGACTTGCACAATATGATGGAAGCTGTCCAAA\nTGCTACAAGGAGTATCACAACAGTTAATTGAACTTGGTAAAGCTACAGTGGGTATAGCGATAGATTTTGA\nTAGGTCACAAAGGAAAATACAATCCTCATTAGGGCTAACTCAAAAGGGTGCAGAGAATCTTGGCAAGATT\nTCAAAAGATGTGTGGAAAAAGGGATTTGGTGAAAGCCTTGAAGAGGTCGATAATTCACTGATTAAAGTCT\nATCAAAATATGCGTGATGTTCCACATGAAGAATTACAAGGGGCATCAGAGAATGTTTTAACATTAGCTAA\nAGTTTATGATGTGGACTTAAATGAAGCAACTCGTGGCGCAGGACAATTAATGTCTCAATTTGGTTTATCT\nACACAACAAACATTTGATTTATTGGCAGCAGGTGCTCAAGCTGGGTTGAACTATTCTGATGAACTCTTTG\nACAATCTTTCAGAGTATGCACCGTTATTCAAACAAGCGGGTTTTAGTGCGGATGAAATGTTTACCATTCT\nTGCGAATGGAACCGCAAATGGATCGTATAACTTGGATTACATTAATGACCTTGTAAAAGAGTTTGGTATC\nCGTGTACAAGATGGTTCTAAAGGTGTATCAGAAGGATTCGGTGATTTATCAGAAGAGACACAAAAAGTAT\nGGGAATCATTCAATGAAGGTAAGGGAACCGCGGCTGCTGTATTTAATGCTGTATTAGGTGACTTACGTAA\nAATGGATGACAAAGTAAAGGCAAACCAGATTGGTGTTGCTCTATTCGGTACCAAATGGGAAGACATGGGG\nGCAGACGCCGTACTAAGTTTAAATGATGTGAATGGTGGTCTGGGTGATGTAAATGGCCGTATGGACGAAA\nTGAAAAAACTGCAAGAAGAATCACTGGGCCAAAAATTCCAGAGCGCATTAAGGGAAACTCAAACAGCACT\nTGAGCCTTTAGGAAAACAACTTGCGGATCTTGCAGCAGATGTTCTCCCTAAAGCTGCAAAAGGACTATCA\nGACCTTGCTGAATGGTTTTCCAAGTTACCAGAACCTGTCCGAAACTTTGTTGCTATTTCTGCCGGATTAA\nCGATTGCTATTACTGCAATTGGCGTTGCTATTGGTGTTTTAACTTTTGCAGTTGGTGCTTTGAATATTGC\nATTAGGACCTGTAATATTAGCGATTTTGGGAATTTCCGCCGCAATAACTATTGCTATAGCGATAGTGAAA\nAATTGGGGTGCTATAACCGATTGGCTTTCCGAAAAGTGGACCCAATTTAAAGATTGGTTTGGTGAATTGT\nGGTCTGGAATAGTTCAGGCTTGTAGTGATGGGTGGTCTTCCACAGTTGAGTACTTTTCCGAAGCCTGGTC\nGTCATTTATTGAAATGATGCATGAATTTTTTGACCCTATAGGTCAGTTTTTTAGTGAATTGTGGTCTGGA\nATTGTCGAAACAGCGTCATCTTGGTGGTCGAATCTTGTCACAACTGCATCAGAATTGTGGAGTCAACTGA\nCTCAAGCCTGGCAAGAAACTTGGAATACGATACTTACCGTCTTAGATCCAATTATTTCGGCGATATCTGT\nCGTTTTAGAAGCAGGTTGGTTATTAATACAGGCAGGTACACAAATTGCCTGGGCCTTAATAAGTAAATAT\nATTATTGATCCGATTACTGAAGCGTACAACTGGTGTAAAAATCAGTTCGGTGAGTTAGTTTCTTGGTTAA\nATTCACAGTGGGAAACGGTAAAATCTTATACACTTGCAGCGTGGAATTTGGTAAAACAGTATGTTATTCA\nACCGGTTCAAGAATTGTGGAATACAACAAAGCAAAAACTTGGAGATTTAGCTAATTGGATACTATCAAAT\nTGGGAATCTATAAAATCCTATACGCTTACGGCGTGGAATTTGGTGAAGAAATATGTGATTGATCCAGTAA\nCTGAAGCCTATAATTCAGCTAAGCAAAAATTTACTGATTTATATAATTCAGCGAAAGAAAAATTCGATTC\nTGTGAAGAATGCTGCACAAGAAAAATTTGATGCTGCTAAACGTAACATCATTGATCCAATAAAAGAAGCA\nGTTGGGAAGGTAGAAGAGTTTGTGGGTAAAATCAAAGGTTTCTTTGATAATTTGAAGCTGAAAATCCCTA\nAACCTGAAATGCCAAAGCTTCCACATTTCAGTCTGCAGACTAGTTCTAAAACAATTGCAGGTAAAGAAAT\nCTCTTATCCATCTGGCATAAATGTGGATTGGCGTGCTAAAGGCGGTATTTTCACACGGCCAACTATTTTC\nGGAATGAATGCTGGAAACTTACAAGGCGCAGGAGAAGCTGGACCGGAGGGGGTTCTACCCTTAAATGAAA\nAGACACTAGGTGCCATTGGAAAAGGAATTGCATCTACGATGACACAACAAAGTAATGACCGTCCAATTAT\nCCTACAAGTGGATGGAAGAACGTTTGCTCAAATCACCGGTGATTATACAGATCATGAAGGTGGAGTGAGA\nATTAGAAAAATCGAAAGGGGGCTGGCATAGATGCTATATGGAATTAATTTTAATGGAAAACATTCATATG\nACGATATGGGATACACTATGCCAGCTGATGGCAGAGATATTGGCTTTCCAAGTAAAGAGAAAATTGTTGT\nAAAAGTTCCTTTTAGTAATGTTGAATACGATTTTAGTGAAATATACGGATCTCAAACATATACTTCAAGA\nACATTGAAATACCAATTTAACGTTTTAAGGCAAGGGAATTTTACACCGCATGCAATGCAAATCGAAAAAA\nCAAAGTTAATTAATTGGCTTATGAATACTGGCGGAAGAAAAAAGCTTTATGATGATACTATTCCTGGTTA\nTTACTTTTTAGCTGAGGTAGAAAGTGCGGCTGATTTCCAAGATGATTGGGAAACGGGAACAATGACGGTA\nACTTTCAGAGCATATCCTTTTATGATTGCTGAATTGTATGAAGGTAATGATATATGGGATAGTTTTAACT\nTTGATTTGGATGTTGCGCAAACTACTAATTTCACTGTGAATGGTATGTTACAAATGGTTTTACTAAATGT\nAGGGGCTTCTGGTGTAGTTCCAGAGATAACAACATCTAAGCAAATGAAGATTATTAAAGATGGAGTCACC\nTATACTGTGTCAACAGGTATTACAAGAGATAAGAATTTTGTACTTAAATCAGGAGAAAATCCCATAAAAG\nTTACTGGTAATGGTACCATCTCATTCCGTTTCTATAAGGAGTTGATTTAAGTGTATAAAGTAACGATTAT\nTAATGATGGTATTGAAACAATAATTCATAGCCCCTATGTAAATGACCTTAAATTACCGTTTGGTGTAATA\nAAAAAAAGTATAAATCTAATAGATGCATTTAATTTTAGTTTTTATATGAATAATCCTGGTTTCCATAAGA\nTAAAGCCGTTAAAAACACTTGTAAATGTGTTAAATACAAAGACAGGTAAGTATGAATTTGAAGGGCGTGT\nGTTAGGTCCAAGTAAGAATATGGACAATTCAGGACTTCATAGTACTTCATATGAGTGTGAAGGTGAACTT\nGGGTATCTACATGATTCAGTTCAACGGCATTTAGAATTCCGTGGAACGCCAAAGGAACTTTTTGCAAAAA\nTTATTGAGTATCATAACAACCAAGTAGAGGAATATAAAAGATTTAAAATTGGAAATGTAACAATTACCAA\nTACTACAAATAACCTTTATCTTTATTTATCAGCGGAAAAAGATACTTTTGAAACAATTAAAGAAAAACTA\nATAGATAAATTAGGTGGCGAACTCCAAATACGTAAAGTAAACGGAGTTCGTTTTTTAGATTATTTAGAAC\nGTGTTGGTGAAGAGAAAAAAACAGAAATTCGAATCGCTAAAAATTTACTCAGTATGTCTTGCGACATAGA\nTCCGACTGAAATTATTACTCGCTTGACTCCTTTAGGTACACGAATAGAGTCAAAAAATGAAGGAGCGACA\nGATGCATCAGAAGCGCGATTAACTATTGAATCAGTTAACAATGGAGTACCTTATATTGATCATCCAAGTG\nGAATAAAAGAGTTTGGTATACAAGGTAAATCTATAACTTGGGACGATGTAACAATAGCAAGTAACTTACT\nTGCAAAAGGAAAAGAGTGGTTTGCAAATCAAAAATCAATTGTAACTCAATATAAACTAAGTGCAGTTGAT\nTTGTATTTAATTGGTCTAGACATCGATTACTTTGAAGTAGGGAATTCTCATCTAGTCATAAATCCTGTTA\nTGGGGATAGATGAGCTTCTAAGGATTGTTGGGAAATCCTTAGATATTAATAATCCACAAGGAGCCAGTTT\nAACAATTGGAGGTGCATTAAAAACATTAAATCAATATCAAAGTGATTTGAAAAAATCAGCACAGCAAGTT\nGTGGATTTACAATATACAGTGCTGAGACAAAACGGGAAAATCAATTCGTTGTCAGCCAGTCTTGTTGAAG\nCAGAAAAATTATTACAGTCCTTAAATGAAGCTGTTGAAAATGCGGATTTACAAATGATTGTTCAGTCAGT\nTTCTGATTTAAAAAGTGCTTTAAAAAAAATTGAAGAGGAAATACAAACTTTACCCACATCCGAAGTGATT\nTTGCAGATGCAAAAAGATATACAAAATAATACAACTGAAATTGTAGATATCGATGAAAGAATGATTGAAT\nCTGAAGTAGTAATTGAAAATAATAGTAAAAGCATTGAAGTATTACAAATCGATTTAAAAGATGTAGTAGA\nTCGTCTAACTGCTTTAGAAAATGGAGGTTCATAATGTGGCAAATATAAGTGGTTTTTTAAATAGAATTCG\nAACCGCTATTTATGGTAAAGATGTTCGTGAGTCTATACATGATGGGATAGAAGCTATCAATAAAGAGACA\nGAGATAGCGACGAAACTTTCCAATAATATAAAGATTAAACAAGTTGCTTTAGAAAAGAAATATGATGACC\nAAATTTCTAACATGACAAATGAAAATCCATCTATTAGTGAATTGGTTGATTTTAGAACTAGTGGATTTAC\nAGGAATGTCATATGTTACAGCGGGAAAAAGAACTGATGCTATTGATGAAAGGATTACAGATATGTCTGTC\nAGCATAAAAGACTTCGGAGCAGACCTCACAGGAGAGACAGACAGTACGGTAGCTATTCAACAAACAATAG\nACTACGTATATGATCGTGGAGGTGGTGTAGTTTATATTCCGCCAGGGATTTTAAGATACACTTCTATAAT\nTGTTAAAATTGGTGTTCATATTCGTGGTTCATTAGTTTCAATTACAGATTGGAATGGGAGGTTAGGCTCT\nAATACGTCCGTAACTACTTTGATACCAACTGATCTTAATAGACCTTCTATCATCATGCATGGGAACTCAA\nCGATTAATGGCGTCTGTTGGTTTTACGAAGGGCAAAAGAAAGAACTGACGTCTGAAAGTGACACATTTAT\nTCAATACCCTCCAACTATTCAGTTGGGTGATGCGTCAAACAAGGCTATTGGAAATTATATTGGAAACTTT\nATAGTAATTGGAGCATATGACTTCATATCGCAATACTCATTTTCAAATAGCGTAGAAAAGTTATTTGTAG\nAAAAAGGATATGGTATGTTCTTAAATACATTTTGTACGCTTAAAAAATGTACTGATATTCCTAGATTCTC\nTAAGATTCATATGAATCTAAATTCAGCATTAGGATGGCTAACTGGAAATACAGTTCCTTATTACTCTAAA\nATCGCTCGTAATGGTGTAATGTTTAAGGTTGCGCGTGTCGATGACTGTGTGATAGAAGATTGTTTTGCAT\nATGGCGTTAAGCACTTTGCTCATCTCTATAAAGAGGTGGGTGAGGATGGGAACGGCGGTGGTATAACAGT\nAATTGGATCAAGCTGTGATGTTTGCCATCAAGCGTTTAGAAACGATCGTGGAAATCTATCGTTTGGCGTG\nAAAGTAATCGGGGGATTTTTCACACCAGTTGTTAACGTGGATGGCAGCGAAAAATGTTTAATTTACCTTA\nGTCAAAACGCTACATATACTAGAATGCAATTTTCGTCATTCAAATGTTATGGAGATGTTGTATCACAGGT\nAAGTAATACTTCAAAAACAGACCATATTGTTATTTTCGAAAACGGAAATGCGGGTAATAACGTCGTAAAT\nTTATTTGGCGATGTACAGTTTAATATTGGTATTTCGAAAAACGAGAACGTGTTGATCAATAATGTTGTAA\nATTTTATTGGTACAGATAGGGATTCCTCTTTATCAAAACTAGATAGAGTGACTATCCGTCATCTTGTAAT\nGACCGGTTCTCGAATTGATCTAACTCATGGTGTCGATGATATTGCATGGTTTAAAGTGAATAATGAATAC\nGCTTTTGGTGCAGTTAAGGATGCCTATGATCCGACATTTTTTACAAAAGCGTATTTCCGACCAGGTACAC\nTGACTTCATTACCAACCGCAAATGCAAATCATCGTGGAAAGATGATTCGTCTGGAAGGTGCAAATGGTGT\nTGAGGATAGGTTATTTATATGTAAAAAGAAAAGCGATGGAACATATCACTGGAAACAGCTAGACGCAGAA\nCAATAATGATGAAGGGGTGTTTTAAATGGGATTGCAAGTTAATATAACTTTATTTAATCAATTAGAAGTA\nAATTCATATGCAAGAGTCGGCAATATTGGAGGAACAAAAGAGGAACAGTTTTTTTCTATTGATTACTACG\nCTTCGCGAAATGCCTTTTTGAGGAAACTTGACCCTATCAAACAAGAAAACTATAAATTCACTCCATCTGT\nTATGGATGACTCATTGAATTTTGTGAAACAAGCTTATATTTATGTTAAAAGAAGAACGGAATTTGCGGGT\nGCTGTCGATGTATTAGAAGAAGGGCAAAAATCTTGAGTTTAATAAAATTCCTCAGTTTATCTAATATATA\nAGATTTTATATGTTATTATATGTAATGGCGTCTTAATATAAGACGCTAATACATATAATTTGGAGGAAAA\nAGATGAACATATCCAGTCACCAAGAGGGAATGTCAAAACTGAATATAATAATACTACTTTTTCTCTCGTA\nTATAATGAGTACTTATAAATACGGGGAATTTGTACATTATTTTGTGTTTATAGTCATGGCCGCTCTTATA\nCTTAGAATGCTTACTGTAGGCATCAAGGTAAGTAATGAAATATCAATAAATAAAGGCATATTGACGTTTT\nGCGTAATTTTATTAGGGTTATTGTTATTGAAATCGATTTTTAACTTAAATGAATTAAAGGCATTTATTAA\nGAATTATTCAGTTATCTATTTCGTTCTAATTTACGGCCTTCTAGAATATTATAAGAATGGTGCTAGGTAT\nGGAGAGCAAGTTTTTATACAGCTTACTAAAATACTAAATGTACTTTCTGTCTTAAACCTAATTCAGGTTG\nTTTTACACAGACCGTTATTATCTGGATTCTTCACAGAACAGATGGACAAGTATCAATATTGGGCGTATGG\nTACTGGTGACTTTAGACCAGTTTCCGTGTTTGGTCACCCTATTGTAAGTGCATTGTTTTTTTCTATATTA\nGTGATCTGTAATCTTTATATACTTAAAGGCAATTTAAAATATCCATTGCAGATTGTGGCACTTGTGAATG\nTCTATGCCTCGCAATCTAGGAGTGCTTGGATTGCATTAGCAATTATTGTATGCTTGTATTTTATTAAAAA\nTTACCGTATTAAAAAAATAAAAAGAAATGTTCGATTTACTTATAGTCAATTATTAAAAATTTATGTTTCA\nTTAGTAATAGTGATCTGTGGATTTGGCTTAGTTGCTTTAAGCTCGGATAGTATCATAAGTTCCATAATTG\nAAAGATTCGGAGATTCTTTAAGTGGGAATAGTAAAGATATTTCAAACTTACAACGGACGGGCACATTAAG\nTTTAATTAATACTTATATGTTTCAACAAGATATGTTTAGATTACTGTTTGGTTATGGATTAGGGACTGTA\nGGAGATTTTATGTCTGTTAATACCGTTGTTATTCGTAATTTCTTGACGACAGATAATATGTATTTGACTT\nTTTTCTTTGAATTAGGGCTCTTGTCATTGATTAGTTATGGATTATTCTTTATTATTGGAGTCATTCGTTT\nTTTCTTATCTAAAAATTACTGGTTAAGCGAATTAGGCGCTTTATGTTTTATCTTTTTATCAATCATAATA\nTTTTTCTTTGAAGGTATAGGGTGGGGTACTGTAGTAACTGTTTGGATGTTTTCTTTACTGACCATTCTAA\nTGAAATTTAAAAATCCAAACAGTTTAACAAAAAATAAAATAAAATAGTTACAGTAGTATTTATACTAGTT\nTTTGGGAGAGAAACATGAACAAAAAACAATTAAAAGAAACAATTAATTCAGACTTATACAGAGTATACGG\nAAGTCAAGGAACAAAAAAGAAAATTATTAATTTCTTAAGAAACCCTGGCTTTAAATATTTATATATACTA\nCGTAAATGTAGTTATTATAAAGAGAAAAACAAGTTAATGTATAGATTGTATTTTATATTATTGTTGCGTT\nATCAGTATAAGTATGGACTAGAAATAATGCCTGATACGAAGATTGGAAAAGGCTTTTACATTGGACATAT\nAGGAGCTATTACAATTAATCCTAAATCTATAATTGGTGAAAATGTAAATATATTAAAAGGGGCATTACTT\nGGCTATAATCCGCGAGGGAAATATAAAGGCTGTCCAACTATAGGAGATGGTGTTTGGATAGGTCCTAATG\nCTGTAATAGTTGGTAATATTACAGTTGGAAATAATGTAGTGGTAGCGCCAAATACGTTAGTCAATAGGGA\nTGTTCCAAGTAATTCAGTTGTGGTAGGTAATCCGTGTCAAATTATTTCACAGGATAATGCAACTGAAGCT\nTATGTCAATTATACAGTTTAACAAAATACAATTTTAGTTGAAAAGGAGACGAGTTATCGTCTTCTTTTTT\nTATTGTCAAAAGGAGATGAGAACAGTGGAAGATGCAATTTTCAATTCAGTCATTCAACAGGGCGCATTCG\nCAGCGTTATTTGTGTGGATGCTATTTACTACACAAAAAAAGAACGAGCAGCGTGAGGAAAAGTATCAACA\nAGTAATTGATAGAAACCAACAAGTAATTGAAGAACAGGCAAAAGCCTTTGGATCTATTTCTAAGGATGTA\nACAGAAATTAAACAAAAATTATTTGAAGGAGATGTTCAATAATGGGATATATCGTTGATATTTCAAAATG\nGAATGGCAATATTAATTGGGATGTAGCTGCAGCGCAATTAGATTTAGTAATTGCTAGGGTACAAGATGGT\nTCAAATGTTGTGGATCATATGTATCAAAGTTATGTGAGTGAAATGAAAAGGCGTGGTGTTCCTTTTGGTA\nATTATGCGTTTTGTCGTTTCGTTTCTGAAAATGATGCAAGAGTTGAAGCGAGGGACTTCTGGAACCGCGG\nTGATAAAGATGCATTATTCTGGGTTGCTGATGTGGAAGTAAAAACAATGGGGAATATGTTAGCCGGAACA\nTTAGCTTTTATCGATGAGTTACGTCGATTAGGTGCTAAAAAGGTTGGTTTATATGTTGGCCATCATACAT\nATAAAGAGTTCCAAGCTGATAAAGTAAACGCTGATTTTGTATGGATTCCTCGATATGGTGGGAATAGACC\nAGCTTATCCATGTGATATCTGGCAATACACAGAGACAGGAAATGTACCTGGTATCGGTAAATGCGATTTA\nAATGAGTTAATTGGTGATAAATCGTTATCTTGGTTTACCAATAATCACATGGGCTTAGTTGTTCCTGAGG\nTAGGAAAGCGAGTTGTTTCTAAAGTAGAGACTCTTAGATTTTATTCAAAACCTTCCTGGGCGGATGTGGA\nTGTTGCTGGAACAGTTTCTAATGGTTTGGGGTTCGCTATCATTGGAAGAGTCGATGTGAATGGTTCGCCA\nCAATATAAAGTACAAAATTCTCGTGGTAGTGTCTTTTATATTACGGCTAGTCCAAAATATGTAGAAGTAA\nAATAAAAATTGCCGGCTCTTAATTGATATGCTCCCCCTATAGTAGACAGAAAAAAGAAAGCTTTATATCC\nTGTCTACTATAAGGGGGATTTTTTTATGGGGAAAATTAGAAGAACATATGATGAAACATTTAAGAAAAAG\nGCTATAGATTTATATTTTAAAGAAGGCATGGGTTATACCCAAATAGGAAAAACACTAAGCATTGATGAAA\nAAAACGTACGTAGATGGGTAAAACGTTTCAAAGAAGAGGGTATCAAAGGCTTAGAAGAGAAACGTGGAAA\nGGCTACTGGAGGAACAAAGGGGCGACCTAAAAATTGTCCAAAGGAGCCTACAGAAAGAATCAAATATTTA\nGAAACAGAGAATGAAATGCTAAAAAAGCTATTGGGAATGTTAAAGGAGGGATGAAAGCTGCACCAGCATA\nCAAAAAATATGAAATCATACAAGAAATGTCCAAAGGTATGCGTTCAATACAGCTACTGTGTAAAATCGCG\nGAAGTATCTAGAAGCGGATATTACAAGTGGTTAAAAAGGCAAAGCAATCCTTCTCCGAAGGAGATAGAAG\nATGAAAAAATAAAGAATAAAATTTTGGAATGTTATAACCAAGTGAAGGGTATCTACGGATACCGAAGAAT\nAACTGTGTGGTTAAAGATGAAACATGGGATCGTCGTAAACCACAAGAGAGTTCAACGACTAATGAATAGG\nATGAAACTTAAAGCAATTATCAGAAAAAAACGACCTTATTTTGTATCAAAAGAAGCATATGTAGTCTCAA\nAGAATTACTTGAATCGAGATTTTAAAGCGGAACAACCAAATGAAAAATGGGTAACAGATATTACTTACCT\nTATTTTTAATGGGAAAAAGCTATATCTATCCGCCATAAAGGACCTTTATAACAATGAAATTGTAGCTTAT\nCATATTAGCCATCGTCATGATATACAACTAGTTATCGATACGCTAAACAAGGCGAAAAAACAACGAAACG\nTGCAAGGGATTCTTTTGCATAGTGATCAAGGTTTCCAATACACTTCACGCCAATATAATGATTTCCTAAA\nAAAACATAAGATGAAAGCTAGTATGTCTAGAAAGGCGAATTGTTGGGACAATGCTTGTATGGAGAATTTC\nTTTAGTCATTTCAAAGCGGAGTGTTTTAATCTGAGTTCATTTCGTTCAGTAGAAGAAGTTAAATTTGCCG\nTACATAAGTATATTCATTTTTATAACCACCATCGTTTTCAGAAAAAATTAAAGAACCTGAGTCCATACGA\nATATCGAACTCAGGCTTCTTAACTATGCGTTTTAATTACTGTCTACTTGACAGGGGTCAGTACATAATTG\nAGTCGGCTTTTTTATTGCAAATCAGATATCCTTCTAACTTGTTTTTCTTTCTTCTTGAAGTTCTTCTTGT\nACTGTTTAAATTCTTCTTTATCTACGTGGAACTTTTCTCCGGTGGCAACGTTTTTAACTAAGTATGTTTT\nGGTTGTAAGAGATTTAATTATGTAATAAATAGCAAATAGCATAAGAGAAATACAAAAGGTAGGAATAGAA\nAATACAATAGCAAGTGCAGCTAGTATATTATCTCTCTTATGATCTCTTTGTAACACTAAACGTTTCCCTG\nCTGCAGCTTGAGCTTGTTCTAATTGTTGCATACGTTGTAGCGATGCTACAGTATCATAACTCATGAAAAA\nACCTCCTTGAAATAATACCTAAATCATACCAATTTCATGTAACAACTGTAAATGTTAGTTTCTCATTTAT\nTGACAAAACAGAACAATTGTTCTACAATTCATTTACAAACAAATGTTCTTGAAAGGGGAAATTCATATGG\nCAGCAACTGAAAATACAACTAAAGGAAAAGAGAAAGCATTAGAAGAGGCTCTGAAGAAAATCGTAAAAGA\nATTTGGTACAGGGGCTATTATGAAACTGGGTGAGCGTCCTAATCAAAAAGTATCTGTCGTATCAAGTGGT\nTCTGTTGGATTAGATAATGCTTTAGGGGTTGGTGGATATCCAAAAGGACGCATTACTGAAATCTTTGGTC\nCTGAATCTTCAGGTAAGACAACTATAGCATTACATGCAATTGCAGAAGCGCAAAAAGAAGGTGGAACAGC\nGGCATTTATTGATGCAGAGCATGCACTTGACCCTATTTATGCACAGAAGTTAGGTGTTAATATTGATGAA\nCTGCTCATGTCACAACCAGATACAGGAGAGCAAGCACTAGAGATTGCAGAAGCTCTAGTTAGAAGTGGGG\nCTGTAGATATCATTGTGATAGATTCTGTAGCTGCTCTAGTCCCGAAAGCGGAAATTGATGGGGACATGGG\nAGATTCACATGTGGGATTGCAGGCTAGATTGATGGGGCAGGCAATGAGAAAAATATCAGGAGCAATATCC\nAAAAATGGAGTTGTAGCAATCTTTTTAAATCAAATCAGAGAAAAAGTTGGTGTTTCTTTTGGGAGTCCTG\nAGACAACACCGGGTGGTAGGGCGTTAAAATTCTATTCAACAATTCGACTAGATGTACGTCGAGGAGAACA\nGTTGAAAGGAAAAGAAAGTGACGTTTTAGGGAATAAAACAAAAGTGAAAGTAGTAAAAAATAAAGTTGCT\nCCACCATTTAAAAACATTGACTTTGACATCTTATACGGAGAAGGGATTTCCCTAGAGGGAGAGCTTATTG\nATATTGGGGTAGAGTTAGATATTGTTCAGAAAAGTGGTGCATGGTACTCGTATCAAGAAGAACGTCTTGG\nGCAGGGTAGAGATAATGCCAAGCAATTTCTGAAAGAGAATGAAAACATACGTAATTCTGTTCGAAATAAA\nATTTATGAATACTATTCTCCAAAAGAAGACTCTGTTGTTGTAAAAGCGGAGCTTATAAAAGAAAATGAGC\nCCATCACTTTAAAAGAATCTGAATAAGGTTATATAAAGAGAATCTTAAAATAAAATTCATGCTATGTATA\nTTTTAAAAACTGGAACACCTAGTTATTAGAACTGGGTGTTTTTATATACTCATAAACTCACGATCAAAAT\nAAAGCTTATCCATTAAGTTTTTTGTATATTATTTTGCAAGTAAGTTCCGTACATATTTACCCAACTGTTC\nATCTGTTAAACCTGACTTAATTGCACCTTTAATAGCATCAATTGTATTAATTGTATGAGGTTCATTCTCT\nTCTTTCTTTTTACTATGTATCCATTTCTTATATAGTTCATCCTCTTTCTTTGCCAAACTCAAAGCGTTCT\nCAACAGTAGTAACTTTCTTCCTCACCCAATGACCAGCTATTTTTTTGACATACTCAGGATTCAATATCAT\nGTCTGATCTCAGCATCACATAATAAATCAAAACATTTATAACAGGGTTAGGCAACCTATAATTATCACGC\nATATCAGTAACTATATGCAAATCCCCTTTTGGAACAGTAGCACCGCCTGATAACTCGCTTAAAAGTTGTT\nCAGGTGTAACAGAATTTAATTGCTGTATCAATTTTTGTTGTTTTTCCATGAATTTATCCTCCTAATACTT\nTGTATAATATAAATTTTATCTAGTAATCTTCCGCATTTAATATAACCCCATTTCATCAGATTCTTCTTTA\nGATAGTTTGTAATATATACTTGGTGCAAGAGGGTTATCTATTGTATTTGTGGAGGGGGTTTTTGAGTCAT\nTATCTTCTAACCAATTATAATATACACTACTAGTATCTGTGTTAGTTTTACGTTTAGAAATATTGTGGAT\nGTCTCTAAATCTATCGATGTAGTCATATACCATACTTTTAAATGTATTAGTAAAGTATCCAATTGGATTT\nTTCATAAGCTCTCCATTGTGTTCAACTTCATGAGTTTTAGAGAATAAAGTAACAGATGCATTCGCAAGGA\nTATTGTTAAATACATCCTTGTCAGATAGTAAACTAAACTTCTTAGCAGCCTTTTTAGCGATATTTACAGC\nATTGTGGAAGGATTCGTTAATAACTTTTGAACCAAATGCAGTTGCTAATTTCATACGCATAGATTGTGGA\nACACGATAGTCGATAAAATCATTATCATTGACTGTAGATTGAAACTCATTTCTATTACGTATATTTATAT\nTTTTTATATTTTGTTTTAAGGTTTTAGTAGTTGTTTTATTGGTGTGACATTTTTCTTCATTTTTAATAGG\nTGCTTGTGTGACAACCTGTTCCTCCACAATAATTGGTTGAATAACAATCGCATTGCATGTTTGCCTCATA\nTCGCTTTTACGCTTCATTTCTAGTTGTTTAATAATACCTAAAGACTCTAAGTGTAGGCATACTCTAATAA\nCCGTTCTACGGCTAATATTGAGGTCTTCAGCGATGTGTTTTTTTGTTCTGAATGATACACCAAAGAACTT\nAGAAGAATAGTTGTGTAATTTATTTAATACAGCCAATTGAGTTTTATTTAACTGATCTACGAATTTTTCT\nTTGTATGCACGAACAGTCTTGTTTAATTCATCGACTGTTTTAAATGTTGCTAGGTTTTCATATGTTTCAT\nTACCTGCAATAATTGTAATTCCTTGTTTCTTTTCCATAACGGTTTGTCTCCTTTTTGGAAACAAAAAAGC\nAACAGAATGCCAAGTGTAAGCAAACTGTTGCTAAGGAGCCCTTACGTACTGTAAAATAGTTCGTAAGAGT\nACAGCAAGTGTTTGCCTAGTGTGATTAGGCGGACGGTATATAGAGTGTTGACGCACTTTATATACACGCT\n\n"class(her1410_1)## [1] "character"dim(her1410_1)## NULLlength(her1410_1)## [1] 1write(her1410_1, file = "her1410_1.fasta")
seq1 <- read.fasta("her1410_1.fasta")As you can see, you can very easily download the data from any NCBI
database. Regarding sequences, the main drawback of the function
entrez_fecth() is that the downloaded sequences are
character objects and transform it into sequences that you can handle
with seqinr or other packages is not straightforward and it
requires some polishing (see ref. 7). Alternatively,
you can save the data as a new fasta file in your computer before
importing with seqinr, as in the following example.
# compare two files
her1410_2 <- entrez_fetch(db = "nuccore", id = her1410$ids[2],
rettype = "fasta")
write(her1410_2, file = "her1410_2.fasta")
seq2 <- read.fasta("her1410_2.fasta")
# codon frequencies
freq1 <- count(unlist(seq1), 3, freq = TRUE)
freq2 <- count(unlist(seq2), 3, freq = TRUE)
stop1 <- freq1[which(names(freq1) == "tga" | names(freq1) ==
"tag" | names(freq1) == "taa")]
stop2 <- freq2[which(names(freq2) == "tga" | names(freq2) ==
"tag" | names(freq2) == "taa")]
stops <- rbind(stop1, stop2)
stops <- as.data.frame(stops)
stops$sequences <- c(her1410$ids[1], her1410$ids[2])
stops <- cbind(stops$sequences, stack(stops[, 1:3]))
names(stops) <- c("sequences", "freqs", "stop_codon")
# plot
library(ggplot2)
p <- ggplot(data = stops) + geom_bar(aes(x = toupper(stop_codon),
y = freqs, col = sequences, fill = sequences), stat = "identity",
position = "dodge", alpha = 0.8)
p + scale_fill_brewer(palette = "Dark2") + scale_color_brewer(palette = "Dark2") +
theme_linedraw() + ylab("Codon frequency") + xlab("Stop codon") +
guides(color = "none", fill = guide_legend(title = "HER1410 sequences"))
Bioconductor
Bioconductor is a repository of R packages for “the analysis and comprehension of high-throughput genomic data”. It started 2002 as a platform for understanding analysis of microarray data, being the first R version 1996, and the first stable beta in 2000. Currently, it contains 2183 packages (release 3.16, November 2, 2022) for diverse bioinformatics tasks. It also contains many learning resources, spanning vignettes, course materials, and workflows.
The diversity of packages in Bioconductor allows working with widely used bioinformatics apps and file formats (fasta, fastq, BAM, SAM, vcf…), for different stages of sequence analysis, quantification, annotation… You can explore packages by categories on the Bioconductor site.
Illustrative description elaborating the different file types at various stages in a typical analysis, with the package names (in pink boxes) that one will use for each stage. From: https://www.bioconductor.org/packages/release/workflows/vignettes/sequencing/inst/doc/sequencing.html
Some of the most common Bioconductor packages and functions are:
GenomicRanges: ‘Ranges’ to describe data and annotation;
GRanges(),GRangesList()Biostrings: DNA and other sequences,
DNAStringSet()GenomicAlignments: Aligned reads;
GAlignemts()and friendsGenomicFeatures, AnnotationDbi: annotation resources,
TxDb, andorgpackages.SummarizedExperiment: coordinating experimental data
rtracklayer: import Genome annotations e.g BED, WIG, GTF, etc.
Annotationhub: access to Ensembl, UCSC, NCBI and other databases.
I recommend you to check the examples in the reference 9 below and other workflows that you can find at Bioconductor.
References
R in action. Robert I. Kabacoff. March 2022 ISBN 9781617296055
Regular expressions in R: https://rstudio-pubs-static.s3.amazonaws.com/74603_76cd14d5983f47408fdf0b323550b846.html
R Programming for Data Science (Chapter 17): https://bookdown.org/rdpeng/rprogdatascience/
A little book of R for bioinformatics: https://a-little-book-of-r-for-bioinformatics.readthedocs.io/en/latest/index.html
rentrez vignette: https://cran.r-project.org/web/packages/rentrez/vignettes/rentrez_tutorial.html
Biostrings: https://kasperdanielhansen.github.io/genbioconductor/html/Biostrings.html
Example of the generation of multiple sequence alignments and phylogenies with R: https://rpubs.com/bhuvic/828540
Where do I start using Bioconductor: http://lcolladotor.github.io/2014/10/16/startbioc/#.Y4ZCaOyZP0p
Bioconductor Workflow: Introduction to Bioconductor for Sequence Data: https://www.bioconductor.org/packages/release/workflows/vignettes/sequencing/inst/doc/sequencing.html
Exercises
Write a function that, given an input name:
1-. It downloads a sequence from the nucleotide database in Genbank. The function needs to check if the different available sequences for that name are identical and rule out duplicates.
2-. It plots the nucleotide composition (% of A, C, G and T) and the amino acids frequencies in the 6 possible frames.
3-. It should also download the protein sequences with that name and count the number of enzymes (you can use ase as keyword) and the number orfans (hypothetical protein).
Test your function with the nucleotide sequences of the plasmids pSE-12228-03 and pCERC7.
Session Info
sessionInfo()## R version 4.2.2 (2022-10-31)
## Platform: x86_64-apple-darwin17.0 (64-bit)
## Running under: macOS Big Sur ... 10.16
##
## Matrix products: default
## BLAS: /Library/Frameworks/R.framework/Versions/4.2/Resources/lib/libRblas.0.dylib
## LAPACK: /Library/Frameworks/R.framework/Versions/4.2/Resources/lib/libRlapack.dylib
##
## locale:
## [1] es_ES.UTF-8/es_ES.UTF-8/es_ES.UTF-8/C/es_ES.UTF-8/es_ES.UTF-8
##
## attached base packages:
## [1] stats graphics grDevices utils datasets methods base
##
## other attached packages:
## [1] ggplot2_3.4.0 rentrez_1.2.3 RColorBrewer_1.1-3 seqinr_4.2-23
## [5] formatR_1.12 knitr_1.41
##
## loaded via a namespace (and not attached):
## [1] pillar_1.8.1 bslib_0.4.1 compiler_4.2.2 jquerylib_0.1.4
## [5] highr_0.9 tools_4.2.2 digest_0.6.30 jsonlite_1.8.3
## [9] evaluate_0.18 lifecycle_1.0.3 tibble_3.1.8 gtable_0.3.1
## [13] pkgconfig_2.0.3 rlang_1.0.6 DBI_1.1.3 cli_3.4.1
## [17] rstudioapi_0.14 curl_4.3.3 yaml_2.3.6 xfun_0.35
## [21] fastmap_1.1.0 withr_2.5.0 dplyr_1.0.10 stringr_1.4.1
## [25] httr_1.4.4 generics_0.1.3 sass_0.4.4 vctrs_0.5.1
## [29] tidyselect_1.2.0 ade4_1.7-20 grid_4.2.2 glue_1.6.2
## [33] R6_2.5.1 fansi_1.0.3 XML_3.99-0.12 rmarkdown_2.18
## [37] farver_2.1.1 magrittr_2.0.3 scales_1.2.1 htmltools_0.5.3
## [41] MASS_7.3-58.1 assertthat_0.2.1 colorspace_2.0-3 labeling_0.4.2
## [45] utf8_1.2.2 stringi_1.7.8 munsell_0.5.0 cachem_1.0.6