Question: one sample from pool shows low quality at the end, why?
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gravatar for CY
9 months ago by
CY270
United States
CY270 wrote:

We have pooled library and do de-multiplex to get fastq for each sample. However, out of these samples, only one sample shows overall low quality at the end of ends. Any possible reason?

fastqc • 305 views
ADD COMMENTlink written 9 months ago by CY270

Hello,

how poor is "poor"? Can you post Screenshots so that we can compare a "good" and a "bad" sample? What sequencing platform was used? How was the library prep was performed? What kind of indexes did you use?

fin swimmer

ADD REPLYlink written 9 months ago by finswimmer7.7k

I am not sure the exact amount of samples. Probably around 10 samples were pooled in the same chip. For one sample, the last 10 bases got quality lower than 30 from fastqc. It was amplicon-sequencing on Hi-Seq.

ADD REPLYlink written 9 months ago by CY270
1
gravatar for genomax
9 months ago by
genomax59k
United States
genomax59k wrote:

This could simply be a bad library that has relatively short inserts which is causing adapter sequence read-through. This generally leads to a rapid drop in Q scores since you start getting low nucleotide diversity.

ADD COMMENTlink written 9 months ago by genomax59k

Thanks. This is a possible reason. I will check the insert size of this sample.

ADD REPLYlink written 9 months ago by CY270

We found that 'N' is enriched at the end of the reads. If this is poor template generation due to low nucleotide diversity? Will the sequence enrich with 'N' or some other phenomenon?

ADD REPLYlink written 9 months ago by CY270

Most likely. When the basecaller loses confidence to call a base it is going to put N in that spot.

BTW: Is the sequence before N's any good or are these just very short inserts or primer dimers? That entire sample may have failed.

ADD REPLYlink written 9 months ago by genomax59k
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