Bad quality at the end of the read 150pb Miseq

[induivain]

Hi everyone!!!

I am working with Sure select capture , and the miseq in a DNAseq experiment.

This is my first time that I use the paired end read at 150bp , and I have a problem at the end of the reads, I have a bias in the last bases. I remove all reads with Q<30 , remove adaptors with cutadapt and trimomatic, but the bias is not resolved.

The mean of the insert size is 198bp , and the adaptors are the tipical for illumina.

The size mean of the library is 350bp before sequecing.

this happens someone???

Thanks in advance.

[MLog]

Hi induivain,

I observe exactly the same effect (even stronger, example attached) on my 250-bp runs. My example is from a recent run of an exome sample, but it seems that this bias is not related to the exome capture, because I see the same on total DNA samples also: http://makarich.fbb.msu.ru/temporary...ce_content.png

[induivain]

Hi MLog,
Thank you for you reply
It is good to know that I am not the only one with this effect.
I am very curious, is posible to know what is the library size before sequencing?

[MLog]

I am very curious, is posible to know what is the library size before sequencing?

Certainly yes - agilent or agarose electrophoresis. (Though actually Agilent sometimes generates weird results). I think this bias could a be result of short inserts, those that are shorter than the read length. In this case you will be reading the adapter sequence. You mention that the insert size is about 200 bp - but this is only the average value. Depending on the distribution of fragment lengths you still can have a number of fragments that are shorter than 150 bp.

[induivain]

You mention that the insert size is about 200 bp - but this is only the average value. Depending on the distribution of fragment lengths you still can have a number of fragments that are shorter than 150 bp.

I was thinking the same , but only 10 % has a minor size that 150bp, and I only use reads between 151bp to 300bp.

Agilent or agarose? , It will be anyone nice.