combining adapters and barcode removal into a single command paired-end fastq

[Grelot]

Hi,

I want to remove adapters and demultiplex by barcode a couple of paired-end fastq files

First this basic command works fine so it means my files are likely to suit with flexbar usage:

flexbar -r $R1_fastq -b $Tags

But when i tried the following command:

flexbar -r $R1_fastq -p $R2_fastq -b $Tags -b2 $Tags -a $Primer_F -a2 $Primer_R -at 0.1 -t 02_demultiplex/ -n 4

• $R1_fastq : a fastq file forward read sequences
• $R2_fastq : a fastq file reverse read sequences
• $Tags : a fasta file with barcode and related tag
• $Primer_F : a fasta file with forward sequence of illumina adapter
• $Primer_R : a fasta file with reverse sequence of illumina adapter

I got the following message error:

               ________          __              
              / ____/ /__  _  __/ /_  ____ ______
             / /_  / / _ \| |/ / __ \/ __ `/ ___/
            / __/ / /  __/>  </ /_/ / /_/ / /    
           /_/   /_/\___/_/|_/_.___/\__,_/_/     

Flexbar - flexible barcode and adapter removal, version 2.5


Local time:            Wed Dec  4 14:16:16 2019

Target name:           02_demultiplex/test/02_flexbar/grinder_teleo1
File type:             fastq
Reads file:            /share/reservebenefit/working/Input_data/Outputs/grinder_teleo1/grinder_teleo1_R1.fastq
Reads file 2:          /share/reservebenefit/working/Input_data/Outputs/grinder_teleo1/grinder_teleo1_R2.fastq   (paired run)
Barcode file:          /share/reservebenefit/working/pierre/eDNA--benchmark_pipelines/02_demultiplex/Tags.fasta
Barcode file 2:        /share/reservebenefit/working/pierre/eDNA--benchmark_pipelines/02_demultiplex/Tags.fasta
Adapter file:          /share/reservebenefit/working/pierre/eDNA--benchmark_pipelines/02_demultiplex/Primer_F.fasta
Adapter file 2:        /share/reservebenefit/working/pierre/eDNA--benchmark_pipelines/02_demultiplex/Primer_R.fasta

threads:               4
max-uncalled:          0
min-read-length:       18

barcode-trim-end:      ANY
barcode-threshold:     1
barcode-match:         1
barcode-mismatch:     -1
barcode-gap:          -9

adapter-trim-end:      RIGHT
adapter-min-overlap:   3
adapter-threshold:     0.1
adapter-match:         1
adapter-mismatch:     -1
adapter-gap:          -6

Barcode:               Sequence:
S1-01                  AACAAGCC
S1-02                  TGAGAGCT
S1-03                  ACAACCGA


[...]
S29-10                 CTTGTGAC
S29-11                 TCTGTTCG
S29-12                 ACCAGTAC

Barcode2:              Sequence:
S1-01                  AACAAGCC
S1-02                  TGAGAGCT
S1-03                  ACAACCGA
[...]
S29-10                 CTTGTGAC
S29-11                 TCTGTTCG
S29-12                 ACCAGTAC

File reading error occured.

Do you have any idea what the problem is?