fastq-multx for dualbarcode-indexed library

[lanc...@genomics.com.tw]

Dear EA Utils, 

I was using the fastq-multx to demultiplex my dualbarocde-indexed library. The output finely demultiplexed into different files based on the barcode list. However, I discovered that only the barcode in the first input file, which happens to be my read1 fastq, were trimmed, but barcode in the second input file, which is my read2 fastq, remained. I was expecting both being trimmed. 

Was it a bug or am I setting the command wrong? 

# my cmd as below 

mkdir -p _test

fastq-multx -b NS14019_barcode_list.txt 1Mtest.R1.fastq 1Mtest.R2.fastq -o ./_test/%.R1.fastq -o ./_test/%.R2.fastq  -m 1 2>./_test/log &

# view my barcode file

cat NS14019_barcode_list.txt

1-16 ATTACTCG-TATAGCCT truseq

2-17 TCCGGAGA-ATAGAGGC truseq

3-18 CGCTCATT-CCTATCCT truseq

4-19 GAGATTCC-GGCTCTGA truseq

5-20 ATTACTCG-AGGCGAAG truseq

6-21 TCCGGAGA-TAATCTTA truseq

7-22 ATTCAGAA-TATAGCCT truseq

8-23 GAATTCGT-ATAGAGGC truseq

9-24 CTGAAGCT-CCTATCCT truseq

10-25 TAATGCGC-GGCTCTGA truseq

11-26 CGGCTATG-TATAGCCT truseq

12-27 TCCGCGAA-ATAGAGGC truseq

13-28 TCTCGCGC-CCTATCCT truseq

14-29 AGCGATAG-GGCTCTGA truseq

15-30 CGGCTATG-AGGCGAAG truseq

# 5' end

cat ./_test/1-16.R1.fastq  |  awk 'NR % 4 == 2' | head -10000 | perl -ne 'print $1."\n" if ($_ =~ /^(\w{8})\w+/)'| sort | uniq -c | sort -nrk1 | head 

   3291 CGAGATGG

   3287 CCCCCTCT

    121 CCCCCTCA

     14 CGAGTTGG

     12 CGAGATGA

      5 CGAGTGGC

      5 CGAATTGG

      5 CCCCTCTC

      4 CCCCCCTC

      4 CCCACTCT

 

cat ./_test/1-16.R2.fastq   |  awk 'NR % 4 == 2' | head -10000 | perl -ne 'print $1."\n" if ($_ =~ /^(\w{8})\w+/)'| sort | uniq -c | sort -nrk1 | head 

   6616 TATAGCCT

     41 AATAGCCT

     33 TAAAGCCT

     16 TCTAGCCT

     15 TGTAGCCT

     12 TATAGCAT

      9 TATAGCCA

      8 TTTAGCCT

      6 TATAGCCC

      6 TATAGACT

Thank you!

[Erik Aronesty]

Normally, with Illumina, the barcode is in a separate file.   I never tested with dual-barcode embedded in sequence.   This is, likely, a bug.

[NGS部張天昫]

Dear Erik, 

Thanks for your reply. 

Are you saying that this program is not suitable for demultiplexing dual-barcoded illumina pair-end data? 

Do you have any suggested program that is capable of this kind of work? 

Thanks again. 

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Lance Chang 張天昫 生物資訊分析專員
Genomics BioSci & Tech. Co., Ltd
Tel: +8862-2696-1658 # 102
Email: lanc...@genomics.com.tw
Address: 4F., No.100, Sec.1, Sintai 5th Road., Taipei County 221
地址: 新北市汐止區新台五路一段92號4樓

[Erik Aronesty]

we have done demux withe dual-barcoded paired end data a lot, just never embedded in the files.

you can run fastq-mcf to trim off the bases in the other file

some modification to the code would be needed to get it to work in one-pass for situations where the barcodes are embeded, but since this situation is very rare, it's never been worked on (i'd be happy to make that change if you really need it, but i'm not doing nextgen stuff full time right now).

On Wednesday, February 25, 2015 at 5:01:40 AM UTC-5, 張天昫 NGS部 wrote:

Dear Erik, 

Thanks for your reply. 

Are you saying that this program is not suitable for demultiplexing dual-barcoded illumina pair-end data? 

Do you have any suggested program that is capable of this kind of work? 

Thanks again. 

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Lance Chang 張天昫 生物資訊分析專員
Genomics BioSci & Tech. Co., Ltd
Tel: +8862-2696-1658 # 102

Eddress: 4F., No.100, Sec.1, Sintai 5th Road., Taipei County 221
地址: 新北市汐止區新台五路一段92號4樓