#install library
#devtools::install_github("ipatop/circRNAshRNAdesign")
library(circRNAshRNAdesign)This package contains ONE function that will generate a DataFrame or tab separated file with the original table with the shRNA oligo design appended.
The input should be a tab sepparated table with circRNA back-splicing junction coordinates and gene names in the format:
Name Chr Start End
This table can have an optional column with the Strand
So far the following species are available:
Fly: dm3, dm6 Human: hs19, and hg38 Mice: mm10 Rat: rn4
The following packages are required, here how to install it
install.packages("dplyr")
install.packages("tidyr")
BiocManager::install("Rsubread")
BiocManager::install("GenomicFeatures")
BiocManager::install("Biostrings")
BiocManager::install("TxDb.Dmelanogaster.UCSC.dm6.ensGene")
BiocManager::install("BSgenome.Dmelanogaster.UCSC.dm6")
BiocManager::install("TxDb.Dmelanogaster.UCSC.dm3.ensGene")
BiocManager::install("BSgenome.Dmelanogaster.UCSC.dm3")
BiocManager::install("TxDb.Hsapiens.UCSC.hg19.knownGene")
BiocManager::install("BSgenome.Hsapiens.UCSC.hg19")
BiocManager::install("TxDb.Hsapiens.UCSC.hg38.knownGene")
BiocManager::install("BSgenome.Hsapiens.UCSC.hg38")
BiocManager::install("TxDb.Mmusculus.UCSC.mm10.knownGene")
BiocManager::install("BSgenome.Mmusculus.UCSC.mm10")
BiocManager::install("TxDb.Rnorvegicus.UCSC.rn4.ensGene")
BiocManager::install("BSgenome.Rnorvegicus.UCSC.rn4")Input should look like this
head(read.delim("../test/circs_totest.txt"))
#> dm6 circ_name SYMBOL Chr
#> 1 chr2R:17275410-17276063 mbl_chr2R:17275410-17276063 mbl chr2R
#> 2 chr3L:24725824-24726292 scro_chr3L:24725824-24726292 scro chr3L
#> 3 chr2R:5423750-5424239 NA_chr2R:5423750-5424239 <NA> chr2R
#> 4 chr3R:4626973-4628349 5-HT2A_chr3R:4626973-4628349 5-HT2A chr3R
#> 5 chr3L:12851309-12852277 Wbp2_chr3L:12851309-12852277 Wbp2 chr3L
#> 6 chr2R:15735091-15737060 CG30089_chr2R:15735091-15737060 CG30089 chr2R
#> Start End
#> 1 17275410 17276063
#> 2 24725824 24726292
#> 3 5423750 5424239
#> 4 4626973 4628349
#> 5 12851309 12852277
#> 6 15735091 15737060Run to create an output table
OligoDesigner(input_coordinates = "../test/circs_totest.txt",output = "../test/New_out.tsv")
#> Loading required package: TxDb.Dmelanogaster.UCSC.dm6.ensGene
#> Loading required package: GenomicFeatures
#> Loading required package: BiocGenerics
#>
#> Attaching package: 'BiocGenerics'
#> The following objects are masked from 'package:stats':
#>
#> IQR, mad, sd, var, xtabs
#> The following objects are masked from 'package:base':
#>
#> anyDuplicated, append, as.data.frame, basename, cbind, colnames,
#> dirname, do.call, duplicated, eval, evalq, Filter, Find, get, grep,
#> grepl, intersect, is.unsorted, lapply, Map, mapply, match, mget,
#> order, paste, pmax, pmax.int, pmin, pmin.int, Position, rank,
#> rbind, Reduce, rownames, sapply, setdiff, sort, table, tapply,
#> union, unique, unsplit, which.max, which.min
#> Loading required package: S4Vectors
#> Loading required package: stats4
#>
#> Attaching package: 'S4Vectors'
#> The following objects are masked from 'package:base':
#>
#> expand.grid, I, unname
#> Loading required package: IRanges
#> Loading required package: GenomeInfoDb
#> Loading required package: GenomicRanges
#> Loading required package: AnnotationDbi
#> Loading required package: Biobase
#> Welcome to Bioconductor
#>
#> Vignettes contain introductory material; view with
#> 'browseVignettes()'. To cite Bioconductor, see
#> 'citation("Biobase")', and for packages 'citation("pkgname")'.
#> Loading required package: BSgenome.Dmelanogaster.UCSC.dm6
#> Loading required package: BSgenome
#> Loading required package: Biostrings
#> Loading required package: XVector
#>
#> Attaching package: 'Biostrings'
#> The following object is masked from 'package:base':
#>
#> strsplit
#> Loading required package: rtracklayer
#> [1] "Generating sequence for circ 1"
#> [1] "Generating sequence for circ 2"
#> [1] "Generating sequence for circ 3"
#> [1] "Generating sequence for circ 4"
#> [1] "Generating sequence for circ 5"
#> [1] "Generating sequence for circ 6"
#> [1] "Generating sequence for circ 7"Run to create a DataFrame, if writetab = F
oligos<-OligoDesigner(input_coordinates = "../test/circs_totest.txt",output = "../test/New_out.tsv",writetab = F)
#> [1] "Generating sequence for circ 1"
#> [1] "Generating sequence for circ 2"
#> [1] "Generating sequence for circ 3"
#> [1] "Generating sequence for circ 4"
#> [1] "Generating sequence for circ 5"
#> [1] "Generating sequence for circ 6"
#> [1] "Generating sequence for circ 7"Output
head(oligos)
#> dm6 circ_name SYMBOL Chr
#> 1 chr2R:17275410-17276063 mbl_chr2R:17275410-17276063 mbl chr2R
#> 2 chr3L:24725824-24726292 scro_chr3L:24725824-24726292 scro chr3L
#> 3 chr2R:5423750-5424239 NA_chr2R:5423750-5424239 <NA> chr2R
#> 4 chr3R:4626973-4628349 5-HT2A_chr3R:4626973-4628349 5-HT2A chr3R
#> 5 chr3L:12851309-12852277 Wbp2_chr3L:12851309-12852277 Wbp2 chr3L
#> 6 chr2R:15735091-15737060 CG30089_chr2R:15735091-15737060 CG30089 chr2R
#> Start End exon_start exon_end length
#> 1 17275410 17276063 chr2R:17275410 chr2R:17276063 653
#> 2 24725824 24726292 chr3L:24725824 chr3L:24726292 468
#> 3 5423750 5424239 chr2R:5423750 chr2R:5424239 489
#> 4 4626973 4628349 chr3R:4626973 chr3R:4628349 1376
#> 5 12851309 12852277 chr3L:12851309 chr3L:12852277 968
#> 6 15735091 15737060 chr2R:15735091 chr2R:15737060 1969
#> seq
#> 1 ATAATGTAAACTCAGCTTACACACAAAAAAGCAGTAAAATATAAAATACAAAAATTAACAGCTGCGTTTATCTATAGATATATATATACACATCTATATATTTGAATAAGCGTTCGATTTGAATCAAAATTATAGCCCAAGCTATCAGCTACCCCCACAAGCTTTGTACTCCACAAAAACGCTAACGAGCAAAATCGATAAAGACCAGATAGCAAATGAAATCATACCATCTGCCATAGATCTATGCTATAAACATATATAGGTGCTGATCTGTGCTCTTGAATTTAGTGTTTTTTGTGTGTGCGTGTCGCGAGAGGAGCACTCAAAACCAAAAAAAAAAAAAAAAACGTATAGTAAAACAAAAAACTATCTCTAGTCGCTAGGCTATAACTATACAAACTCCAGCTATCCCAGATCCAGATCCGTAAACAGCAAATAGTTATATATCTATAACCCCAAAATACGATGGCCAACGTTGTCAATATGAACAGCCTGCTCAACGGCAAGGATTCGCGCTGGCTGCAATTGGAGGTCTGTCGCGAGTTCCAGCGCAACAAATGCTCGCGCCAGGACACCGAATGCAAGTTCGCCCATCCCCCGGCCAACGTGGAGGTCCAGAACGGCAAGGTCACCGCCTGCTACGACAGCATCAAG
#> 2 CGATAAAAGCCTACTACCAACTTGTAAAACAATTGTTACGCATGTCATCGCACGGCCTTGCTTACACAACAAGAATTGAAAGAAAGTCTTATAGAGAACTACAAATCAATAGAGATCAATACTTTGTAACTGCGCCAAATGAAGAAGATTTGGTTATGAGTTTATCTCCAAAGGACACACTTATACATACTGCCATTTCCCAGCACCATCAAGTGGATACTTCTACTAAATTAAATACAAACGAAACATCGACACAAAATACCGTATCTACAGCAGCCGCGGCAGCGGTTGCACATCACCACCACAACCTTTCTAGTATTCACCACCTCCAAAACCTGCATAGTCAGCATCAAAGTACTTTATTTAATAGTAATCACTCAACACCCTTTAGCGTGACCGATATCTTAAGTCCAATTGAAGAATCGTATCGCAAACTGGAACTGAACGGAAATCCACCATCTCCGTTTCG
#> 3 AAAATCAACCAGATCTTACACCAATGGGGTCCTTCTGGCAGCAACATGTGGCCACTCCTAGTAGTTCAACTCCATTCGATTCACCATTTTTTTCGGCAACACACGGCCTTGTGCAGACTCACCCGTCTATAGGTGGAAGCCACTCTGGGCCCGCTCCATTAAATGGCAATAGCCTTGGGTCTGGAGCTCCATCCTCAATCCTAGCTGGTACTAAGCCAGCCCTAACCGCTGGAAGCGGTTACCTAAACTCACGCGGGAGTCTACCGACCTCTGCACGTTATCCAGCTAATAAGATCACAGGAACCGTCGTGGAACCCAACCCAAAATCTCCATTCCGCCACTTAGATTTCTCTACTAGTGCAACTGCCGAGCTGCGGCGCAATCCTGCGCTATCTGCTCCTGATGAATGTGCACGCGCTTGTCGCGAAGGCGAGCCTCCGAGAATTTGCTACTATCATTTTACGTTGGAATACTACACAGTATTGGGAGC
#> 4 GGTATTGGCCACTTGGCTTTACCTGGTGCAACATTTATGTGACGTGTGACGTACTGGCCTGCTCATCCAGTATATTGCACATGTGCTTCATCAGTTTGGGACGCTATATGGGAATAAGAAATCCATTGGGCTCGAGGCATCGATCTACGAAACGATTGACTGGTATAAAGATAGCCATTGTTTGGGTAATGGCCATGATGGTATCCAGTTCAATAACCGTTCTTGGTCTGGTCAATGAGAAGAACATAATGCCCGAGCCTAACATATGCGTTATCAACAATCGCGCCTTTTTCGTGTTTGGATCTCTGGTAGCTTTTTATATACCCATGCTGATGATGGTTACCACATACGCATTGACGATTCCCCTCCTCCGGAAGAAAGCGCGATTTGCCGCCGAGCACCCGGAAAGTGAACTTTTTCGCAGGTTGGGTGGACGCTTCACCCTAAGGCCGCAGCACAGCCAGCAGCAGTTGCAGATGTTCAGTAGCTTTTCTGGCAGCAATAACAAATTCCTATCAATGGGCGACGGCAATCGCAACTTCAACACTGAAGGAGAGATGGAGGAGGGTGGAGCTCCTAGCAGAAGGAGTGGCGTAGAGCCCGCCGAACGACCTTTGATGCAACAGCGAACGGCGAGCAGCAGGAGCATGGGCACGGTTAGTTTCCGTAATGTCGTGAACGGGACTAGCGGAGCCGCAGGAAGTGGGCGCAGGACGACTGGCACTGCCCACAGCAGCTTCCGGTTCTCTGGCGCCGGCATCTTTCGGCACTCGTCGTCGTCTCCCGCCTCGAGCTGCCACTCCACCAGCACGTCGCGTTCGAGCTCCTTCTGGCGCAAACACGGCGGCTATCCAAACCTAATGGACAG
#> 5 CACCACCTGCTCACCAACCAGCACCGCAACAGCCGCAGGCACCGTCGTCGAATGAACCCAATTGGTATGGTTTTTCAGCGCCGCCGCCGCAGCAGCAACAACAGCAGCCGGGCTATGGGCCACAAGGATGGGCCGGTGGCTATGTCCAACCTCCGCCGTACGCTTCATGTGCCCCAAATTGTCCACCACAGCCGCCGTACGCCGCTCCGGCTCAGACGTACAATGGTCCACAGCCATATGGTGGATACGGCAATGTTCCTGGCGGATTTAATACCCCCGGATTTCAGCAACCGAATGGAAATCCAAATGGCTATCCAGGTGGACCACCGCCACCCGGACAACAAGCAGCAGGAGCAGCTGGAGGAGCCGCCGCATCTGGAGCCAGCTTTATGGGCTTCAGCTTGCCCCCTGGAACAAAAGATAAAAAAATATGAGCAAAAGGACAAGAAAATTATGAACATCTTTAAAATAGTCGAAACATCTAAAGATCTAAAATGTTATCAAGAATTTAATTGAAGTATCATGAACCACCTTTTGCTTTCGGACGCTCAAAGTATTCAAGAGGCATCCAAAAACCATTCAGAAGCATTCGTTCTGTTTATTTTTCTCACTTACAAAATTATTCAATTTGTCCAAATAATAAATGATTTGTTAGCATAATTCTGTAATTAAAAGCAAACTACTGTACCTCAAATAGCCATCCCGACAA
#> 6 AATTGCGATCCATTCACGCCAGCAACTGCAACGCAGCACAACAGCAACACTGAGACAACGGCAACGGAAACACAAAGCCCAGCAACGAATGCAAGATCACAACAAAGCGAGAAAAACCCAGGCAGCAGCGCAGCGAATTGCATTATCAATGAACAGAGAGATGAGGCAGCAACTGCAACAGCGACATCAGCAGCAGCAGCTGCAGCAGCGGCAGCAGGGGCAGGGAATGAGGAAACCAGCTCGGTGGCAGTGCAGGTCAGTCAACCTGTTTCCTGTACCGCCAGAGAGCGTAGAGAGCTGCTGAGAGCCAGCAACACGAACACGCCAGCGAGCAGCAGCAGCAGCACCAGCAGCGCCAGCAACATTAGCTACGCGAGCAACATCAGCGGCGAGCGCTCAGCGATCAGTCTCTCGGCAACTTTCGGCGGGCCAACGCTGTACAGCGCGAGCAGCAGCAACCGGAATTGCTACAACAGCAGCAGCAGCAGCAGCAACTATCGCCCACTGTACGACGATTCGAGTAGCAGCAGCGTGCTGCGGCCCAACAAATGTTTGGCAGAACTCGAGCGTCTCTATGCCGAATTTCGCGCGAGTGAAAGCAGATTCGAACGGGGTCTGATCCAGTGCCAGTTGGACATTGAAAACGCCAACGCCACCGACACCGTTAGCGAAATGCGTCTAAATGTGCCAGCCACTGCGGCAGCAGACACTGTTGCTAGTTGTAGCAACAGCAACAGCAGCAGCAACAACAACAGCAGCAGCAGCAGCAGTGTGTTCCTCAATGTGTCCGGCAGCAGCAGCACCGATAGCGGCGACAATCAAGTTGTCGATGAGGCTAGAAGCATTAGCAACATTGCTGTGCTGCCAGCAACAGCAACTGCAACAACAACAACAGCCACTTCCAAGTCCTGCCAGCGACAGCCGAGCCTAACGATCCAAGTGAACAACAACAACAACAACAACAACAATAACAACGGCAACACTAACGGTACTAGTAACGGCAATCGGGTCTCGGTGGCTTCGCCCACGAGCATTGTGAACAACAACAACGGCGTGTGCGTTGTCAACTGCAGCTACACCCCCAGCAGCAGCAGCAGCAACAACAACAGCAGCAGCAGCAGCAGCAGCTGCATCAGCAACACCACCAACAGCAACATCAGCAACAATAACAGCGTGGTTGAAATCAATAAGTGTGTGCCAGCAGCAGCCAAAGTGTTTAGTGCAAGTGTTCAAAATCAAAATAGGCTCAATAATAATAGTAGCTGCCCAAAAGGGGATCAGCAACATGTTGCTGCTGCATCCACGGCCAGCAATAATAATCATGTGCAGCCTGATAACAGTGTGAATATAATCAGTGTGAATGCAAGCAGCAGCAGCTACAATAATAACCACCCACGCACCTTCACCTCAACGGAATGCCAAACGGATGATCTGTCAGCCAGTACATCATCCAACATGCAACAGCAACACCAGCAGCAGCAGCAGCAGCAACAGATCCGGACACGGGAGCAACGTCGCAAGGAGCGGCGGGAACGCCGGCAGCAGCAACAGCAGCAACAACAGTTTCAGCCACACCATGCGCGACGCCACCCGCCCCCACCTCCTCCGCCACAACTACACCCACCACCGCATCTGCACCACCCACATCCCCACCCACTTCCCCACCCTCATCCGGCTGCCATGGGACTGGCACGCGCTCTGTTGCCCGACATCCTGCACGCCCACTACCCGCCGCCCTATACTGCTCTGCCCGTGCCCGTGGCCATGTCCGCCGCTCCGCCACCGCCGCCCCCGTCGACTGCCGTTGCGGCATCTGCTGTTGCGGCGGCGGCACAGCAGCCAGCACATGCAAGTCCGGCTCCTCCGCCAGCGCTGACGTCCGTGATATCGACGGTGCCACTGCCGGGTCCGCTGCCAGCGCCTCTCATGAACGACGGACGCTTCACGCTGCCGCTGCCCATCATGCGCAG
#> Strand junction junction_revcomp
#> 1 1 CAGCATCAAGATAATGTAAAC GTTTACATTATCTTGATGCTG
#> 2 1 CTCCGTTTCGCGATAAAAGCC GGCTTTTATCGCGAAACGGAG
#> 3 1 TATTGGGAGCAAAATCAACCA TGGTTGATTTTGCTCCCAATA
#> 4 1 TAATGGACAGGGTATTGGCCA TGGCCAATACCCTGTCCATTA
#> 5 1 ATCCCGACAACACCACCTGCT AGCAGGTGGTGTTGTCGGGAT
#> 6 1 TCATGCGCAGAATTGCGATCC GGATCGCAATTCTGCGCATGA
#> TopStrand
#> 1 ctagcagtCAGCATCAAGATAATGTAAACtagttatattcaagcataGTTTACATTATCTTGATGCTGgcg
#> 2 ctagcagtCTCCGTTTCGCGATAAAAGCCtagttatattcaagcataGGCTTTTATCGCGAAACGGAGgcg
#> 3 ctagcagtTATTGGGAGCAAAATCAACCAtagttatattcaagcataTGGTTGATTTTGCTCCCAATAgcg
#> 4 ctagcagtTAATGGACAGGGTATTGGCCAtagttatattcaagcataTGGCCAATACCCTGTCCATTAgcg
#> 5 ctagcagtATCCCGACAACACCACCTGCTtagttatattcaagcataAGCAGGTGGTGTTGTCGGGATgcg
#> 6 ctagcagtTCATGCGCAGAATTGCGATCCtagttatattcaagcataGGATCGCAATTCTGCGCATGAgcg
#> BotStrand
#> 1 aattcgcCAGCATCAAGATAATGTAAACtatgcttgaatataactaGTTTACATTATCTTGATGCTGactg
#> 2 aattcgcCTCCGTTTCGCGATAAAAGCCtatgcttgaatataactaGGCTTTTATCGCGAAACGGAGactg
#> 3 aattcgcTATTGGGAGCAAAATCAACCAtatgcttgaatataactaTGGTTGATTTTGCTCCCAATAactg
#> 4 aattcgcTAATGGACAGGGTATTGGCCAtatgcttgaatataactaTGGCCAATACCCTGTCCATTAactg
#> 5 aattcgcATCCCGACAACACCACCTGCTtatgcttgaatataactaAGCAGGTGGTGTTGTCGGGATactg
#> 6 aattcgcTCATGCGCAGAATTGCGATCCtatgcttgaatataactaGGATCGCAATTCTGCGCATGAactg
#> junction5p junction_revcomp5p
#> 1 CATCAAGATAATGTAAACTCA TGAGTTTACATTATCTTGATG
#> 2 CGTTTCGCGATAAAAGCCTAC GTAGGCTTTTATCGCGAAACG
#> 3 TGGGAGCAAAATCAACCAGAT ATCTGGTTGATTTTGCTCCCA
#> 4 TGGACAGGGTATTGGCCACTT AAGTGGCCAATACCCTGTCCA
#> 5 CCGACAACACCACCTGCTCAC GTGAGCAGGTGGTGTTGTCGG
#> 6 TGCGCAGAATTGCGATCCATT AATGGATCGCAATTCTGCGCA
#> TopStrand5p
#> 1 ctagcagtCATCAAGATAATGTAAACTCAtagttatattcaagcataTGAGTTTACATTATCTTGATGgcg
#> 2 ctagcagtCGTTTCGCGATAAAAGCCTACtagttatattcaagcataGTAGGCTTTTATCGCGAAACGgcg
#> 3 ctagcagtTGGGAGCAAAATCAACCAGATtagttatattcaagcataATCTGGTTGATTTTGCTCCCAgcg
#> 4 ctagcagtTGGACAGGGTATTGGCCACTTtagttatattcaagcataAAGTGGCCAATACCCTGTCCAgcg
#> 5 ctagcagtCCGACAACACCACCTGCTCACtagttatattcaagcataGTGAGCAGGTGGTGTTGTCGGgcg
#> 6 ctagcagtTGCGCAGAATTGCGATCCATTtagttatattcaagcataAATGGATCGCAATTCTGCGCAgcg
#> BotStrand5p
#> 1 aattcgcCATCAAGATAATGTAAACTCAtatgcttgaatataactaTGAGTTTACATTATCTTGATGactg
#> 2 aattcgcCGTTTCGCGATAAAAGCCTACtatgcttgaatataactaGTAGGCTTTTATCGCGAAACGactg
#> 3 aattcgcTGGGAGCAAAATCAACCAGATtatgcttgaatataactaATCTGGTTGATTTTGCTCCCAactg
#> 4 aattcgcTGGACAGGGTATTGGCCACTTtatgcttgaatataactaAAGTGGCCAATACCCTGTCCAactg
#> 5 aattcgcCCGACAACACCACCTGCTCACtatgcttgaatataactaGTGAGCAGGTGGTGTTGTCGGactg
#> 6 aattcgcTGCGCAGAATTGCGATCCATTtatgcttgaatataactaAATGGATCGCAATTCTGCGCAactg
#> junction3p junction_revcomp3p
#> 1 CGACAGCATCAAGATAATGTA TACATTATCTTGATGCTGTCG
#> 2 CATCTCCGTTTCGCGATAAAA TTTTATCGCGAAACGGAGATG
#> 3 CAGTATTGGGAGCAAAATCAA TTGATTTTGCTCCCAATACTG
#> 4 ACCTAATGGACAGGGTATTGG CCAATACCCTGTCCATTAGGT
#> 5 GCCATCCCGACAACACCACCT AGGTGGTGTTGTCGGGATGGC
#> 6 CCATCATGCGCAGAATTGCGA TCGCAATTCTGCGCATGATGG
#> TopStrand3p
#> 1 ctagcagtCGACAGCATCAAGATAATGTAtagttatattcaagcataTACATTATCTTGATGCTGTCGgcg
#> 2 ctagcagtCATCTCCGTTTCGCGATAAAAtagttatattcaagcataTTTTATCGCGAAACGGAGATGgcg
#> 3 ctagcagtCAGTATTGGGAGCAAAATCAAtagttatattcaagcataTTGATTTTGCTCCCAATACTGgcg
#> 4 ctagcagtACCTAATGGACAGGGTATTGGtagttatattcaagcataCCAATACCCTGTCCATTAGGTgcg
#> 5 ctagcagtGCCATCCCGACAACACCACCTtagttatattcaagcataAGGTGGTGTTGTCGGGATGGCgcg
#> 6 ctagcagtCCATCATGCGCAGAATTGCGAtagttatattcaagcataTCGCAATTCTGCGCATGATGGgcg
#> BotStrand3p
#> 1 aattcgcCGACAGCATCAAGATAATGTAtatgcttgaatataactaTACATTATCTTGATGCTGTCGactg
#> 2 aattcgcCATCTCCGTTTCGCGATAAAAtatgcttgaatataactaTTTTATCGCGAAACGGAGATGactg
#> 3 aattcgcCAGTATTGGGAGCAAAATCAAtatgcttgaatataactaTTGATTTTGCTCCCAATACTGactg
#> 4 aattcgcACCTAATGGACAGGGTATTGGtatgcttgaatataactaCCAATACCCTGTCCATTAGGTactg
#> 5 aattcgcGCCATCCCGACAACACCACCTtatgcttgaatataactaAGGTGGTGTTGTCGGGATGGCactg
#> 6 aattcgcCCATCATGCGCAGAATTGCGAtatgcttgaatataactaTCGCAATTCTGCGCATGATGGactg