demultiplexing Illumina output with fastq_multx
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5.8 years ago
Floydian_slip ▴ 160

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 • 3.5k views
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Were you able to solve your problem? How did you do it?

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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.

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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.

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