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Old 10-12-2018, 01:13 AM   #1
Location: Germany

Join Date: Apr 2018
Posts: 14
Default Do my FASTQC results indicate sequencing bias?

I used the Illumina Nextera Flex kit to sequence bacteria (WGS) and I got two warning that I usually do not get when using Nextera XT:

1. overrepresented sequences in the first read (R1) but not the second read (R2).

2. warning/error k-mer content

Why do I get such errors? Does this indicate sequencing bias?
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File Type: pdf FASTQC.pdf (100.3 KB, 30 views)
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Old 10-12-2018, 07:24 AM   #2
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Location: East Coast USA

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This data must have been sequenced on NextSeq (or NovaSeq). That string of "G" is probably no-signal which is interpreted as G base.

The bias at the beginning of the reads is likely due to the Nextera transposase sequence bias (similar to bias seen in "random priming") .

If these are the only two "issues" then your data looks ok. Proceed with your normal analysis.
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Old 10-14-2018, 09:44 PM   #3
Location: Germany

Join Date: Apr 2018
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Thank you for helping me!
We use a MiseQ and it was the first time that we used Nextera Flex instead of XT. And it`s also the first time we got such a string of G.

Beside these issues, I also got a warning for "per base sequence content". However, using Nextera XT always gives such issues.
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