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.
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Originally posted by Allison Harmon View PostNo. 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.
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
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Originally posted by kmcarr View PostAllison,
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.
Jon
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Originally posted by JBKri View PostHow 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
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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?
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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).
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Originally posted by JBKri View PostHow 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
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Originally posted by Catherine_Burke View PostHas 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?
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:
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.
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Originally posted by AKrohn View PostYou 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
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