Best approach for targeted PCR resequencing on MiSeq from RNA

[Dessi]

Hi everyone,

I need to amplify selected regions from my RNA samples and resequence with MiSeq.

I am unclear to what is the best approach:

1. Use the Illumina metagenomic 16s sequencing library kit, design PCR primers with the Illumina adapter sequences attached as part of the primers. Pool the amplicons. Then I use the Nextera index kit to index the samples.

or

2. Perform my own PCR independent of Illumina kits, purify the PCR fragments and then add adapter and index sequences.

Advice on multiplexing PCR software is also highly appreciated!

Regards,

Dessi

[luc]

There is no need to buy the Illumina indexing kit. These are just simple PCR primer oligos and you can have them synthesized elsewhere according to your needs. The sequences are in the 16S protocol and the indices in the Illumina adapter letter.

[Dessi]

Thanks Luc,

I have 8 samples and 5 or 10 amplicons in each sample. It would be easier for me to pull the PCR products of each sample together and then index them. However, I am confused to each kit to use.

A second concern I have is about the low complexity library sequencing. I have been reading on forums that sequencing libraries from a few amplicons only might be an issue.

Cheers,
Dessi

[nucacidhunter]

In this experiment there would maximum of 8x10=80 amplicons. There are two options for library prep:

1- Set up PCR reactions with standard primers, clean up and pool amplicons from each sample and follow ligation based library prep.

2- Follow Illumina’s 16S two step PCR protocol. In this case every target from each sample can be amplified separately with overhang primers followed by clean up and pooling each sample amplicons and then adding index with 2nd PCR using Nextera primers.

As there are around 10 different amplicons, low diversity should not be an issue. Just keep in mind that amplicons sizes should be suitable for intended length of reads.