Question: demultiplexing Illumina output with fastq_multx
2
gravatar for nbhardwaj
19 months ago by
nbhardwaj80
United States
nbhardwaj80 wrote:

Hi, I am running fastq_multx to demultiplex Illumina files. I have 2 paired-end files: Undetermined_S0_L001_R1_001.fastq.gz and Undetermined_S0_L001_R2_001.fastq.gz

They use different combinations of i5 and i7 barcodes/indices for R1 and R2 paired-end files. Here is the index info from the SampleSheet.csv (tab-separated): Sample_ID i7 Sequence i5 Sequence NA12877_A1 A707 CCCAACCT A505 CTAATCGA NA12877_A2 A708 CACCACAC A505 CTAATCGA NA12877_A3 A709 GAAACCCA A505 CTAATCGA NA12877_B1 A710 TGTGACCA A505 CTAATCGA NA12877_B2 A711 AGGGTCAA A505 CTAATCGA NA12877_B3 A712 AGGAGTGG A505 CTAATCGA NA12878_A1 A707 CCCAACCT A506 CTAGAACA NA12878_A2 A708 CACCACAC A506 CTAGAACA NA12878_A3 A709 GAAACCCA A506 CTAGAACA NA12878_B1 A710 TGTGACCA A506 CTAGAACA NA12878_B2 A711 AGGGTCAA A506 CTAGAACA NA12878_B3 A712 AGGAGTGG A506 CTAGAACA

So, this is the tab-separated barcode file I prepared: id seq style NA12877_A1 CCCAACCT-CTAATCGA TruSeq NA12877_A2 CACCACAC-CTAATCGA TruSeq NA12877_A3 GAAACCCA-CTAATCGA TruSeq NA12877_B1 TGTGACCA-CTAATCGA TruSeq NA12877_B2 AGGGTCAA-CTAATCGA TruSeq NA12877_B3 AGGAGTGG-CTAATCGA TruSeq NA12878_A1 CCCAACCT-CTAGAACA TruSeq NA12878_A2 CACCACAC-CTAGAACA TruSeq NA12878_A3 GAAACCCA-CTAGAACA TruSeq NA12878_B1 TGTGACCA-CTAGAACA TruSeq NA12878_B2 AGGGTCAA-CTAGAACA TruSeq NA12878_B3 AGGAGTGG-CTAGAACA TruSeq

I am expecting 12 paired-end fastq sets. I run fastq_multx in the following way:

fastq-multx -B barcodes.txt Undetermined_S0_L001_R1_001.fastq.gz Undetermined_S0_L001_R2_001.fastq.gz -o %_R1.fastq -o %_R2.fastq

All the reads are assigned to Unmatched_R1 and R2.fastq files with no reads going to the sample files which are all 0 in size.

What am I doing wrong? Is my barcode file wrong?

Thanks!

demultiplexing • 1.1k views
ADD COMMENTlink written 19 months ago by nbhardwaj80

Were you able to solve your problem? How did you do it?

ADD REPLYlink written 8 days ago by Dataman240
1

I actually could not solve the problem. If you find the solution, please let me know too. I ended up using the de-multiplexing performed by the MiSeq Reporter itself such that MiSeq directly wrote individual FASTQ files.

ADD REPLYlink written 8 days ago by nbhardwaj80

Sure, I will let you know! However, it seems that I am also going to use the MiSeq Reporter for de-multiplexing even though I have not had time to figure it out how. I assume that I should make a sample sheet file and in the sample sheet I should set the workflow to be 'de-multiplexing' or something like that. I have tried it on BaseSpace by making a sample sheet with Application,FASTQ Only in the header but it did not work for some reason.

ADD REPLYlink written 1 day ago by Dataman240
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1571 users visited in the last hour