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No index in the undetermined fastq file

The data was generated in MiSeq , demux was done in LRM but it didn't yeild any data assigned to the sample, but the other issue is in the undermined files we are not able to see in any index in the header of the undermined fastq files, which seems bit strange.

Any suggestion how to troubleshoot ,since SAV looks all fine the index read have good Q30 scores as adding the information index read info

zcat Undetermined_S0_L001_R1_001.fastq.gz | head @M06594:100:000000000-DN78N:1:1101:16087:1338 1:N:0:0 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTNTNNNNNTTTTTTTTTNNNNNNNNTTTTTTTTNNTTTTTTTTTTTT + 1111>1>10000AA//AA/>///////<<<</<//-------:-9-9-9-999-9--9-9-9-9-------9-9-999-9-9--9------9-----99--9---#:#####99--9----########--999---##9----9----99 @M06594:100:000000000-DN78N:1:1101:15529:1349 1:N:0:0 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTNNNNNNNNTTTTTTTTTTTTTTTTTTTTTT + 11111>1>>>>0///AA/////>////<<//////--<<-----9-9--9---9----99----9-999--999-------99//999:-9-99-------999----;---9--------########-----;---99-----9---9- @M06594:100:000000000-DN78N:1:1101:15215:1371 1:N:0:0

Any suggestion how to troubleshoot, what could be the issue?

bcl2fastq
• 1,704 views • link
written by 1769mkc 1769mkc

2 answers

we are not able to see in any index in the header of the undermined fastq files, which seems bit strange.

If phiX was spiked in then that is not indexed. So the undetermined reads can at least partly be phiX. Rest could be "junk" data as they all appear to be poly-T in example you show.

• 0 views • link
written by GenoMax GenoMax

yes 15% phix was spiked in, now since that was in LRM i did try in bcl2fastq i can see the index in the header but no difference in the output . I did try swapping the index, then Reverse compliment the i5 index but no difference as such.

• 0 views • link
written by 1769mkc 1769mkc

Run bcl2fastq or bcl-convert yourself, and the indices might turn up.

• 0 views • link
written by swbarnes2 swbarnes2

that what i did it was a samplesheet issue it was resolved it seems in the LRM the samplesheet which was used having some issue.

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written by 1769mkc 1769mkc

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