Amplicon sequencing: Method to prevent end over-representation & improve uniformity

[ECO]

Thought people would be interested in this!

Biotechniques. 2009 Mar;46(3):229-31.

Method for improving sequence coverage uniformity of targeted genomic intervals
amplified by LR-PCR using Illumina GA sequencing-by-synthesis technology.

Harismendy O, Frazer K.

Scripps Genomic Medicine, Scripps Translational Science Institute, Scripps
Research Institute, La Jolla, CA, USA.

One approach for high-throughput population-based sequencing of targeted intervals in the human genome is to amplify the regions using long-range PCR (LR-PCR) followed by sequencing with next-generation sequencing (NGS) technologies. Utilizing this method, we have observed that the 50 bp located at the amplicon ends account for more than 50% of the sequenced bases and that the sequence coverage depth of base pairs within an amplicon is highly variable. Here we propose an explanation for the overrepresentation of the amplicon ends and show that the use of 5'-blocked primers for the LR-PCR reaction reduces their
overrepresentation. Furthermore, we demonstrate that using a 600-bp library insert size rather than the standard 200-bp insert size results in more uniform sequence coverage depth. The capability to increase sequence coverage uniformity greatly improves the effective throughput of NGS platforms.

PMID: 19317667 [PubMed - in process]

[Michael Smith]

Does this apply to 454 also?

Interesting way to even out coverage across LR amplicons by blocking the ends on the PCR products. It works for solexa. What about 454?

[Nitrogen-DNE-sulfer]

It should work with 454
we've done this on SOLiD as the amines block T4 DNA ligase so the ends can't ligate to the adaptors.
I'm curious if the 600bp insert size holds on other platforms?
Any library amplification of molecules that large can create GC bias and I've not seen alot of slxa papers with this size?

[Fad2012]

Hello guys

If the fragmentation is following a normal distribution, shouldn't the ends be underrepresented (less coverage or the rest of the amplicon). But what is appearing in this paper is the opposite?