Metagenomics (not 16s!)

[microgirl123]

Our core facility has many people (with very little money) interested in doing metagenomics on the MiSeq, but they often have different regions of interest. So we're looking for a different way to approach library prep other than the Caporaso approach of using PCR primers that include primers for the region of interest, indices, and Illumina adaptor sequences. This way seems to work well, but is very cost prohibitive since you need to order a separate reverse primer for every sample you want to multiplex. If you only want to do this once, you are paying ~$200 or more per sample for primers. For a set of 20 samples this can add up!

Has anyone seen or used a method that just ligates the Illumina adaptors/indices onto PCR amplicons? This would allow us to place the problem of actually amplifying the DNA onto our customers (and this is a big deal!) and should drastically save on the cost of library prep for these types of samples.

Thanks!

[GW_OK]

We've made libraries out of amplicons for a few customers. Seemed to work ok. Just treat the amplicon like a pre-sheared sample.

[koadman]

Hi microgirl123,
One way to reduce the primer cost is to use the dual-barcode capabilities of the miseq. You can modify the Caporaso et al 2012 oligos to have a barcode at both ends. I have had successful runs using the following adapter structure:

Fwd primer:
AATGATACGGCGACCACCGAGATCTACAC [Barcode] TATGGTAATT GT GTGCCAGCMGCCGCGGTAA

Rev primer:
CAAGCAGAAGACGGCATACGAGAT [Barcode] AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT

This structure is compatible with the standard miseq dual-index program, e.g. read 1, index 1, index 2, read 2. The standard miseq program uses 8 base barcodes but you could get away with fewer. You would need O(N^0.5) barcodes for N samples.

[Vinz]

I guess what you are looking for is the 4primer approach: You have outer primers including your indexes and inner primers with your target specific sequence. Outer and inner primers have an overlap. That way you can reuse the outer primers for independent projects and the inner target specific primers are less expensive (~60bases).
Look in this thread for design of dual indexing:

http://seqanswers.com/forums/showpost.php?p=76937&postcount=13

These are the parts that we use successfully as overlap regions:
ACACTCTTTCCCTACACGA
GTGACTGGAGTTCAGACGTG

[bstamps]

We designed our primers to have a barcode built into our forward primer- since it only added 12bp to a 15bp primer the cost per-primer wasn't that bad. End results from the run were good...forward read was of high quality, with some issues on the reverse. Still we had enough sequence to do what we needed to do with our amplicon. We had some ideas passed to us from our sequencing provider on how to fix this the next time that we're going to try out. Having a barcode on the reverse as well could help as well (Increase in cost).

[microgirl123]

We've made libraries out of amplicons for a few customers. Seemed to work ok. Just treat the amplicon like a pre-sheared sample.

When you say to treat it like a pre-sheared sample, do you mean that you shear your amplicons?

We don't want to do that, but I'm wondering if I can just take amplicons that are ~500 bp and ligate on Illumina adapters and indices. Since they're PCR amplicons, shouldn't they already be A-tailed? Otherwise, we could run them through a TruSeq kit for end-repair, A-tailing, and adapter ligation, but adjust the final size selection to keep the right-sized pieces.

[microgirl123]

I guess what you are looking for is the 4primer approach: You have outer primers including your indexes and inner primers with your target specific sequence. Outer and inner primers have an overlap. That way you can reuse the outer primers for independent projects and the inner target specific primers are less expensive (~60bases).

Thanks! That might work - we can have customers deal with the inner primers for their region of interest and we just take their PCR amplicons and PCR on the outer primers.

[bstamps]

When you say to treat it like a pre-sheared sample, do you mean that you shear your amplicons?

We don't want to do that, but I'm wondering if I can just take amplicons that are ~500 bp and ligate on Illumina adapters and indices. Since they're PCR amplicons, shouldn't they already be A-tailed? Otherwise, we could run them through a TruSeq kit for end-repair, A-tailing, and adapter ligation, but adjust the final size selection to keep the right-sized pieces.

By pre-sheared I think he means your amplicon is already to an insert size that is acceptable to library prep- all fragments are roughly the same size (It being amplicon), so all you need to do is ligate the sequencing adaptors on and go. No need to shear, just prep and sequence.

[microgirl123]

Hi Vinz, do you have any papers or protocols that use this method? It seems like it would be the best approach for us - we'd just need to figure out how to do the actual final PCR (it seems like you wouldn't need many cycles) and how much DNA from the first PCR to ask for in the first place.

[Vinz]

We actually include the outer primer in the target specific PCR. However, the PCR should not be tricky. I would do a 1:10 dilution and 5 cycles. I am not aware of a publication even though it probably has been published.
Fluidigm documentation includes hints on that approach. Good luck!

[GW_OK]

By pre-sheared I think he means your amplicon is already to an insert size that is acceptable to library prep- all fragments are roughly the same size (It being amplicon), so all you need to do is ligate the sequencing adaptors on and go. No need to shear, just prep and sequence.

That's precisely it. It'll be the tightest size selected peak you'll ever make a library out of.

[tickseq]

dual indexing

Thanks for posting your dual indexing primer sequences. I was planning on using the Nextera barcode sequences but wanted to double check. The Illumina rep said that the posted sequences are the sequences as read by the MiSeq. Does that mean that the 5'-3' barcode sequences on the primer should all be reverse complemented from what Nextera provides?

Also, do you have any feelings on standard desalted primers vs HPLC or PAGE purified primers? I've found that people have strong feelings either way but would like to get away with the cheapest method that works.

Thanks

Hi microgirl123,
One way to reduce the primer cost is to use the dual-barcode capabilities of the miseq. You can modify the Caporaso et al 2012 oligos to have a barcode at both ends. I have had successful runs using the following adapter structure:

Fwd primer:
AATGATACGGCGACCACCGAGATCTACAC [Barcode] TATGGTAATT GT GTGCCAGCMGCCGCGGTAA

Rev primer:
CAAGCAGAAGACGGCATACGAGAT [Barcode] AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT

This structure is compatible with the standard miseq dual-index program, e.g. read 1, index 1, index 2, read 2. The standard miseq program uses 8 base barcodes but you could get away with fewer. You would need O(N^0.5) barcodes for N samples.