Insert size on PE 2*250

Question

[David]

Hi, I´m sequencing the DNA of a bacterial genome with illumina (2x250 PE).

I have cleanup reads and mapped them back to a reference genome for that bacterial that is close at 99%.

! Qualimap insert size distribution

I have an expected size distrubution around 300, i was expecting higher insert size , around 350 ?? Do you think it´s ok ?

[David]

Thanks for the update.

I´m using bwa to map reads back to the reference and view bam with IGV. I found strange that i have no reads mapped to the plasmid of the reference (althought the reference contains a plasmid in the reference.fa file). However if i copy only the plasmid to another file (let say plasmid.fa) and map the reads to the plasmid only i recover most of it.

How it comes that in the first mapping of reads against the reference genome + plasmid i do not have any reads that map to the plasmid (no secondary alignments, nothing) ??

Thanks,

ps: Guess that 2*150 bp is a much better idea !!!!

[GenoMax]

Please use ADD COMMENT/ADD REPLY when responding to existing posts to keep threads logically organized.

Answer 1

[piet]

In most cases these libraries with average insert size much smaller than read length assemble well for most part of bacterial genomes. But it is a hugh wast of effort, with most of your fragments you will sequence a long tail of nonsense. And you will loose the ability to assemble accross some (shorter) repeats, which means you will get more contigs than with larger insert size.

i was expecting higher insert size , around 350

Why do you expect higher insert size? The recommended insert size for 2x250 is well above 500 nt. Insert size depends on the protocol you use for library preparation. In future, you could either change you protocol, or stop sequencing after 150 cycles.