[Allison Harmon]

No. The protocol you refer to targets regions V3 and V4 using 2 x 300 cartridges. I want to yarget region v4 using 2 x 250 cartridges. Illumina has an application note on this subject, but it offers few details. I am looking for a more detailed protocol than whats on the application note. Thanks though.

[nucacidhunter]

A good option is to follow Illumina protocol that DRYTCYV is reffering and add Illumina adapter overhang nucleotide seauences to V4-specific sequences instead of V3-V4 region.

[kmcarr]

No. The protocol you refer to targets regions V3 and V4 using 2 x 300 cartridges. I want to yarget region v4 using 2 x 250 cartridges. Illumina has an application note on this subject, but it offers few details. I am looking for a more detailed protocol than whats on the application note. Thanks though.

Allison,

There are two published protocols for targeting just the V4 region (515f-806r).

The first is from Rob Knight's lab:

Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Lozupone, C. A., Turnbaugh, P. J., et al. (2011). Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proceedings of the National Academy of Sciences, 108 Suppl 1, 4516–4522. doi:10.1073/pnas.1000080107

And the second from Patrick Schloss':

Kozich, J. J., Westcott, S. L., Baxter, N. T., Highlander, S. K., & Schloss, P. D. (2013). Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Applied and Environmental Microbiology, 79(17), 5112–5120. doi:10.1128/AEM.01043-13

We have successfully used both protocols in our lab.

[JBKri]

Allison,

There are two published protocols for targeting just the V4 region (515f-806r).

The first is from Rob Knight's lab:




And the second from Patrick Schloss':



We have successfully used both protocols in our lab.

How do you mix the custom primers (Read1, Read2, Index) into the primer reservoirs (wells 12-14)? We find it difficult to avoid introducing bubbles during mixing, and we have not yet found a pipette tip long enough to reach the bottom of the well.
Jon

[kmcarr]

How do you mix the custom primers (Read1, Read2, Index) into the primer reservoirs (wells 12-14)? We find it difficult to avoid introducing bubbles during mixing, and we have not yet found a pipette tip long enough to reach the bottom of the well.
Jon

I don't do the lab part, but those that do tell me that they remove the primer mixes from their MiSeq cartridge reservoir using a sterile pasteur pipet, transfer each mix to a separate 1.5ml eppendorf tube. The needed custom primers are added to each, mixed and then the mixture is returned to the appropriate well in the cartridge.

[Catherine_Burke]

Has anyone used the Caporaso protocol with the V3 600 cycles kits? We have previously used the V2 500 cycles kits successfully, but when we tried it with the V3 600 cycle kit, we got really low quality reads for index 1. We're wondering if the synthesis of read 1 through the index 1 region might interfere with the index 1 read.

Has anyone else had any experience with this?

[jdelton]

Catherine we regularly use a similiar approach (see appendix D for primer design) http://www.mothur.org/w/images/0/0c/..._MiSeq_SOP.pdf with V3 2x300 reads. We run up to 500bp fragments (not counting the primer sequence as that is not part of the read in this approach). The read 2 in the v3 is fairly noisy so you will need to be sure not to use too long of a fragment as stitching will be very difficult (in fact we don't use Mothur as it is not able to stitch together this size fragment without swamping your RAM - we use QIIME and PEAR). If you want to get the highest quality read, then full overlap will be the best approach, but if you need a longer fragment then this approach is very effective. We will occasionally push the limits of this approach, but some common 454 primer combinations (530F-1100R and ITS1-ITS4) are simply too long and either won't stitch at all or we lose 1/2 of the organisms (e.g.Ascomycota are able to be stitched together but Basidiomycota are not).

[LVAndrews]

How do you mix the custom primers (Read1, Read2, Index) into the primer reservoirs (wells 12-14)? We find it difficult to avoid introducing bubbles during mixing, and we have not yet found a pipette tip long enough to reach the bottom of the well.
Jon

You probably already figured this out, but I just use a 10uL pipette. Bubbles aren't going to make a bit of difference here. Pipette the primer to the side of the well where you can see it. Then use the "slosh method" to mix your primer in with the rest of the fluid. Works great every time. Others I know use a Pasteur pipette instead, but this is inaccurate. From a 100uM stock, you should only need 3uL, but I know people who use as much as 15uL. I use 5uL and all is well. Just tap the reagent cartridge against the bench to send everything downward and you should be fine.

[LVAndrews]

Has anyone used the Caporaso protocol with the V3 600 cycles kits? We have previously used the V2 500 cycles kits successfully, but when we tried it with the V3 600 cycle kit, we got really low quality reads for index 1. We're wondering if the synthesis of read 1 through the index 1 region might interfere with the index 1 read.

Has anyone else had any experience with this?

I have run the 515/806 products on 300, 500, and 600 cycle kits. It is no different, but as jdelton says, read 2 in the 600 cycle kits is noisy. Schloss posted some data somewhere that he tried showing to Illumina (they didn't care) that pretty much shows the reagents still peter out after cycle 500. Generally I put the 16S amplicons on 600 cycle runs when also doing ITS amplicons for the same samples and I want the extra read depth. However, the 300 cycle reads will read through your 16S products so be sure to remove the primers.

Method 1: fastx_trimmer, cut the sequences to 150 bases. Not very elegant. 150 is arbitrary, but no primer contamination

Method 2: fastq-mcf (ea-utils), much better. Include all of the possible reverse complemented sequences for 515 and 806 (2 sequences for 1, 18 sequences for the other due to degeneracy). enter this way:

fastq-mcf -0 primerfile.fa read1.fq read2.fq -o read1.mcf.fq -o read2.mcf.fq

Then run the mcf-treated data through your joining program. I just use fastq-join for this. I don't like the qiime wrapper because I lose the stderr output which is interesting to me. You can borrow my joining workflow if you like from our lab website here:

http://nau.edu/Merriam-Powell/EnGGen/Information-and-Resources/

Scroll down to "monsoon resources" (monsoon is our compute cluster), and click on the PE read joining workflow. There is a separate one depending on if you have single or dual indexed data. It will take you to a google spreadsheet. Download as excel or save to your own google drive. Then enter information as appropriate in the first page and take the code you need from the "bash script" worksheet. Not exactly elegant, but much faster and easier than typing a bunch of commands in , and no waiting between steps.

[JBKri]

You probably already figured this out, but I just use a 10uL pipette. Bubbles aren't going to make a bit of difference here. Pipette the primer to the side of the well where you can see it. Then use the "slosh method" to mix your primer in with the rest of the fluid. Works great every time. Others I know use a Pasteur pipette instead, but this is inaccurate. From a 100uM stock, you should only need 3uL, but I know people who use as much as 15uL. I use 5uL and all is well. Just tap the reagent cartridge against the bench to send everything downward and you should be fine.

Thank you; I did not think "sloshing" would be sufficient! I found some long syringe needles that I used for mixing, and the run went well, except the cluster density was really low
J

[RCJK]

You can get ART 200ul extended length pipette tips that fit nicely into the wells and reach the bottom. Really good for mixing the primers in without the need for any sloshing.