Question: Targeted NGS: amplicon length vs read length
1
gravatar for Luca Beltrame
4.5 years ago by
Luca Beltrame220
IRCCS Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy
Luca Beltrame220 wrote:

Hello,

In our group we are about to design an amplicon-based targeted panel for NGS, and we're aiming at around 150 bp for mean amplicon sizes. The wet lab people came back to me asking to calculate the optimal number of samples to be loaded in the instrument (MiSeq). And this is where I have my question: ideally I'd like to pick 2x150bp as read length, so I can have a greater output, however the mean amplicon size might be close to the read length, or in some cases, lower.

Hence my question: would it be "safe" to use a read length approximately equal to the amplicon length so that it doesn't complicate my analysis job afterwards (I'm thinking about adapter read through)? My previous experience always had a larger amplicon length (> 200 bp) so it can't really compare.

Should I still go for 2x150bp, or aim lower at 2x75bp? If I use 2x150bp, would possible adapter read through throw a spanner in the works, or should soft clipping (from BWA) be enough?

sequencing next-gen amplicon • 3.2k views
ADD COMMENTlink modified 4.5 years ago by WouterDeCoster44k • written 4.5 years ago by Luca Beltrame220

The MiSeq software can remove the adapter if you give it in the sample sheet. We don't really see related downstream problems.

ADD REPLYlink written 4.5 years ago by Zaag800

We don't use the MiSeq software, everything is done off-instrument.

ADD REPLYlink written 4.5 years ago by Luca Beltrame220

There are plenty of tools to use after sequencing, not required to use the MiSeq software for that.

ADD REPLYlink written 4.5 years ago by WouterDeCoster44k

How do you run the MiSeq then?

ADD REPLYlink written 4.5 years ago by Zaag800

You can specify whether you want data to go through variant calling on the machine or not. The on board alignment and variant calling is what I would consider "OK." I also do all of my analysis on other machines so I can run different variant calling algorithms, etc.

ADD REPLYlink written 4.5 years ago by DG7.1k
2
gravatar for DG
4.5 years ago by
DG7.1k
DG7.1k wrote:

I'd still do 2x150 if the amplicon targets are 150bp on average. You can deal with the adapter read through and trimming fairly easily. Something like 200bp averaged amplicons might be a better size overall, but either way I think is fine.

ADD COMMENTlink written 4.5 years ago by DG7.1k
1
gravatar for WouterDeCoster
4.5 years ago by
Belgium
WouterDeCoster44k wrote:

Try to cover your amplicons completely in both directions, gives a higher confidence in your variant calling. Adapter trimming is easily to achieve, using e.g. Trimmomatic.

ADD COMMENTlink written 4.5 years ago by WouterDeCoster44k
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