Amplicon based sequencing on the MiSeq

[nvpatkar]

Dear Users
I am an absolute newbie on NGS and wish to sequence about 25 genes on the MiSeq using an amplicon based approach for library preparation. I have some doubts and i hope that someone will help

I would like to ask some questions on targeted gene sequencing which utilizes an amplicon based approach for library preparation (about 250 amplicons).
I wish to synthesize primers from IDT or similar company. Cost is an concern so I would like to use this approach.
My question pertains to designing the primer. I understand that the design of the forward and reverse primer would include the illumina sequencing adapter (p5, p7), barcode and the actual forward or reverse target primer.
I have the adapter sequences from illumina.

My questions are as follows:

1: Do the barcodes have to be present only on both the forward and reverse primers, or just the reverse is OK?
2: What is the permissible length of the insert (given that 120bp for both the adapters, 30bp for both the target sequence primers). And with that logic what is the maximum allowable length of the entire amplicon (400bp, 450bp?) for the illumina MiSeq platform (given that max read length on MiSeq is 250bp)
3:When you design these amplicons, should they overlap and if so by how many bases?

I am looking forward to your reply. Like I said, I am a newbie so please excuse me if I have said something silly

Nikhil

[mcnelson.phd]

Hi Nikhil,

1. There are a couple of ways that you can go about this, but the easiest is to essentially replicate how TruSeq works and only use one index. This would be present on your reverse (read 2) primer so that it's sequenced immediately following read 1. A good idea of how to properly design the primers is to look at some of the 16S amplicon papers that use the TruSeq design as they've been used quite a bit now. Some people have tried making their own dual-index primers that follow how Nextera works, but that's more difficult and most people don't need the extra throughput that allows.

2. The maximum total length for an amplicon (sequence of interest + adapters) on the MiSeq is about 1Kb. That's a physical limitation limit that an Illumina rep once told me as a rough guide for maximum insert length. We've been working with 400bp insert lengths for TruSeq libraries on our MiSeq and they work well. For Nextera libraries we've had library sizes ranging up to 800bp that have also worked well.

3. If you want to overlap your reads, using 2x250, you'll unfortunately need to shoot for around a 400bp insert size and expect a lot of data to get thrown out. Illumina has a software issue that currently causes read quality to nose-dive if the library doesn't have an even base composition (which is a problem with amplicons). They're working to solve that issue, but there's no timeframe for when the fix will come out. Even with genomic libraries you'll see that quality isn't as high over the last 50-100 bases, which can have a negative effect on your analysis.

As an additional note: be prepared to spend a lot of time getting your primers to work properly. Primers ~80nt long really screw with amplification efficiency.

[nvpatkar]

Hi there, many thanks for your reply! I sure did need that perspective

[SeqR&D]

Illumina sells primers with P5/P7 and the indeces and the adapters. So, to save money, the length of your primers only need to have the locus specific sequence and enough of the index primer sequence. You can also do this PCR in one tube. 4 primers, 1 tube. The primers can have an index on one or both sides, and you can choose to read none, one or two.

[james hadfield]

Fluidigm's access array prptocol explains how to accompish this pretty well. Ask Illumina TechSupport for the sequence information letter and you should be able to add tails to your locus specific primers for a secondary PCR that will add on the indexes and adapters you need for sequencing.