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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?
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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. -
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.Will I miss out on information because the insert size (350bp) is larger than the total sequencing read length (300bp).
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.Should I always make the insert size smaller than the read length?
It will sequence 75 bases from either end of insert fragment.What if I wanted to run a 2x75bp read length , how much will it sequence of the 300bp insert size?Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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