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seqgirl123 06-25-2014 05:16 PM

insert size for sequencing
 
I have a genome library at 350bp. I sequenced it using 2x150bp read length on Miseq v2. Will I miss out on information because the insert size (350bp) is larger than the total sequencing read length (300bp). Should I always make the insert size smaller than the read length? What if I wanted to run a 2x75bp read length , how much will it sequence of the 300bp insert size?

gaoch 06-25-2014 06:36 PM

The truth is that reads are overlapped, so the missed part of this read pair should be revealed by another one. Besides, a library with insert size of 350bp should have a size around 350, may be 250~450 sometimes. furthermore, sequencing 250 bp insertion with 2*150bp, I would like to say it's a waste of data, cause there are 50 bp is sequence twice.

nucacidhunter 06-25-2014 07:42 PM

Quote:

Will I miss out on information because the insert size (350bp) is larger than the total sequencing read length (300bp).
It depends on what you are going to use the reads. If you wanted to overlap the paired end reads (R1,R2) to produce longer ones, you will not be able to do it for most of the reads. But, if you are using them to assemble longer contigs most likely it will not make any difference.

Quote:

Should I always make the insert size smaller than the read length?
As explained above if you want to merge read1 and read 2 of fragments you will need to have some overlap in the middle and your insert should be smaller than total cycle numbers.

Quote:

What if I wanted to run a 2x75bp read length , how much will it sequence of the 300bp insert size?
It will sequence 75 bases from either end of insert fragment.


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