fastq_multx help

[n.bha...@celmatix.com]

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: 

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 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.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!