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XmaX 09-20-2021 07:02 AM

Do I need to use a library preparation kit with MiSeq?
Hello everyone.

I am very new to NGS (and to libraries in general) and so I have a few basic questions.

I am planning an experiment in which I will have a number PCR reactions with the intention of introducing single point mutations in the product (PCR products without adapters would be 130bp). Different conditions will be tested, and so I will have 10 different samples of randomly mutated PCR products which I will then sequence using MiSeq. All reactions will use the same amplification primer and template, and so every read would ideally only differ by 1bp.

The first question is whether I need to use a library preparation kit at all? My initial plan was to simply do the mutagenesis-PCR reaction, followed by another PCR round to add indexes, run samples on gel, do gel extraction of my bands, pool the products in equal amounts and proceed with denaturation and dilution. However, I now see the Nextera XT protocol which looks like it adds a lot of steps, and more importantly, doesn't work with PCR products <300 bp. Can I therefore just use an indexing primer kit (does it matter much if it's from Illumina, NEB or anywhere else)?

Secondly, is there a huge advantage of using pair-end instead of single-read in my case?


luc 09-24-2021 05:14 PM

You will not need a library prep kit. You can add all the required Illumina sequences by PCR (in one round or two rounds). This might give you an idea: https://dnatech.genomecenter.ucdavis...umina-systems/
Your libraries will have a very low complexity by default. Thus, you will need to sequence your amplicon pool together with PhiX or another complex library.

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