Issue with Demultiplex dual indexed Illumina reads using bcl2fastq, for beginners.

Question

[salvatore.digiorgio]

Hi everybody!

I am not experienced with sequencing, so maybe there might be a simple solution to the following stuff. We ran a sequencing run on an Illumina NextSeq 500 this week with 100 cycles and dual indices.

This is our samplesheet :

[Reads]
100
100

[Settings]
Adapter,CTGTCTCTTGATCACA

[Data]
SampleID,SampleName,SamplePlate,SampleWell,I7IndexID,index,I5IndexID,index2,SampleProject,Description
1,A21A1tail,DNAsamples,A01,N701,TAAGGCGA,S501,GCGATCTA,,
2,A22A1liver,DNAsamples,A02,N702,CGTACTAG,S501,GCGATCTA,,
3,A25A1tumor,DNAsamples,A03,N703,AGGCAGAA,S501,GCGATCTA,,
4,B31E63Atail,DNAsamples,A04,N704,TCCTGAGC,S501,GCGATCTA,,
5,B32E63Aliver,DNAsamples,A05,N712,GTAGAGGA,S501,GCGATCTA,,
6,C41AR6tail,DNAsamples,A06,N706,TAGGCATG,S501,GCGATCTA,,
7,C42AR6liver,DNAsamples,A07,N707,CTCTCTAC,S502,ATAGAGAG,,
8,C43AR6tumor,DNAsamples,A08,N708,CAGAGAGG,S502,ATAGAGAG,,
9,F31RA7tail,DNAsamples,A09,N701,TAAGGCGA,S502,ATAGAGAG,,
10,F32RA7liver,DNAsamples,A10,N702,CGTACTAG,S502,ATAGAGAG,,
11,F33RA7tumor,DNAsamples,A11,N703,AGGCAGAA,S502,ATAGAGAG,,

And this is my runinfo file:

 <Reads>
  <Read Number="1" NumCycles="100" IsIndexedRead="N" />
  <Read Number="2" NumCycles="8" IsIndexedRead="Y" />
  <Read Number="3" NumCycles="8" IsIndexedRead="Y" />
  <Read Number="4" NumCycles="100" IsIndexedRead="N" />
</Reads>

I wanted to demultiplex our dual index data and then align the obtained fastq files with BWA. Issues occurred with bcl2fastq, I run it with following command line:

bcl2fastq -R /tank/External_HDD/181024_NB501608_0106_AH32KVAFXY/ -o /tank/DONATI/ -i /tank/External_HDD/181024_NB501608_0106_AH32KVAFXY/Data/Intensities/BaseCalls/ --sample-sheet /tank/External_HDD/181024_NB501608_0106_AH32KVAFXY/NS3152197-REAGT.csv -r 30 -p 30 -w 11

Is it correct to obtain 8 fastq files for each sample? How I have to proceed to align them? I appreciate if you advised me some manual i can study from.
thank you.

[GenoMax]

Is it correct to obtain 8 fastq files for each sample?

No. You should only get 2 files (R1/R2) per sample.How did you get 8?

[salvatore.digiorgio]

This exactly is what I expected

Answer 1

[GenoMax]

Ah you got lane specific files (L001 through L004 in names). Since there are 4 lanes on a NextSeq flowcell (not physical but optical) you have 8 files (4 x R1/R2).

You could re-run bcl2fastq with --no-lane-splitting option to just get 2 files per sample.