Question: De-multiplexing Single Index BCL & Fastq reads?
0
gravatar for Jon17
2.8 years ago by
Jon1710
Jon1710 wrote:

I have a mix of single index and dual index Illumina NextSeq reads mixed with dual index. The program bcl2fastq demultiplexed all of the dual index reads great, but I can't seem to figure out how to demultiplex single index reads. Suggestions / help wanted please! I'm interested in learning how to demultiplex single index from both / either bcl and / or fastq files. So any solution will be appreciated!

Undetermined_S0_L001_R1_001.fastq.gz
Undetermined_S0_L001_R2_001.fastq.gz
Undetermined_S0_L002_R1_001.fastq.gz
Undetermined_S0_L002_R2_001.fastq.gz
Undetermined_S0_L003_R1_001.fastq.gz
Undetermined_S0_L003_R2_001.fastq.gz
Undetermined_S0_L004_R1_001.fastq.gz
Undetermined_S0_L004_R2_001.fastq.gz

This is my SampleSheet.csv. Everything from sample 250 on worked great. Everything from 61-25 not so much

Sample_ID,Sample_Name,Sample_Plate,Sample_Well,I7_Index_ID,index,I5_Index_ID,index2,Sample_Project,Description
61,,,,,CGATGTAT,,NNNNNNNN,,
62,,,,,TGACCAAT,,NNNNNNNN,,
63,,,,,ACAGTGAT,,NNNNNNNN,,
115,,,,,GTGGCCAT,,NNNNNNNN,,
116,,,,,GTTTCGAT,,NNNNNNNN,,
117,,,,,CGTACGAT,,NNNNNNNN,,
166,,,,,ATCACGAT,,NNNNNNNN,,
167,,,,,TTAGGCAT,,NNNNNNNN,,
168,,,,,ACTTGAAT,,NNNNNNNN,,
220,,,,,TAGCTTAT,,NNNNNNNN,,
221,,,,,GGCTACAT,,NNNNNNNN,,
222,,,,,CTTGTAAT,,NNNNNNNN,,
247,,,,,AGTCAAAT,,NNNNNNNN,,
248,,,,,AGTTCCAT,,NNNNNNNN,,
249,,,,,ATGTCAAT,,NNNNNNNN,,
250,,,,,ATTACTCG,,AGGCTATA,,
253,,,,,ATTACTCG,,GCCTCTAT,,
254,,,,,TCCGGAGA,,GCCTCTAT,,
255,,,,,CGCTCATT,,GCCTCTAT,,
251,,,,,TCCGGAGA,,AGGCTATA,,
274,,,,,CGGCTATG,,GCCTCTAT,,
275,,,,,TCCGCGAA,,GCCTCTAT,,
276,,,,,TCTCGCGC,,GCCTCTAT,,
277,,,,,AGCGATAG,,GCCTCTAT,,
278,,,,,ATTACTCG,,AGGATAGG,,

ADD COMMENTlink modified 2.8 years ago by h.mon29k • written 2.8 years ago by Jon1710
0
gravatar for genomax
2.8 years ago by
genomax84k
United States
genomax84k wrote:

Look into --use-bases-mask option for bcl2fastq.

To get only those samples that contain the single index you would use --use-bases-mask Y*,I6,n6,Y*. You will have to edit RunInfo.xml file in flowcell folder (make a backup first) and change the line <Read Number="3" NumCycles="6" IsIndexedRead="Y" /> to <Read Number="3" NumCycles="6" IsIndexedRead="N" />. Edit the SampleSheet.csv to remove all 2D samples. When you run bcl2fastq do not forget to specify a new location for output directory.

ADD COMMENTlink written 2.8 years ago by genomax84k

Actually, there is no need to edit RunInfo.xml. You can do everything with --use-bases-mask alone.

Also, do not use Ns as an index.

ADD REPLYlink written 2.8 years ago by igor10k

Thanks igor, so it should look like this?

61,,,,,CGATGTAT,,,,

ADD REPLYlink written 2.8 years ago by Jon1710

Yes. As long as you left the right number of , that match number of fields in the header.

ADD REPLYlink written 2.8 years ago by genomax84k

2D = dual index? Assumptions are the mother of all screwups....

ADD REPLYlink written 2.8 years ago by Jon1710

That is correct. The 2D part for sure :)

ADD REPLYlink modified 2.8 years ago • written 2.8 years ago by genomax84k
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