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Old 04-14-2016, 10:26 AM   #1
sme.bug
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Default Metabarcoding coverage question

Hello,

I want to use MiSeq to metabarcode arthropod samples. I am producing 300 bp amplicons from 130 samples with approximately 600 specimens per sample. This will yield 78,000 300bp amplicons that I want to sequence. Assuming I have the same number of amplicons from each specimen, is it reasonable to expect I will detect most of them?

Because I have 300 bp amplicons, to calculate coverage do I simply divide the number of reads produced in a 2x300 MiSeq run by the number of specimens I want to sequence?

I am still learning the basics of NGS and am unclear on the difference between clusters and reads, among other things.

Thank you for any insights.
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Old 04-14-2016, 12:51 PM   #2
GenoMax
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Experimental experts (informatics person myself) would offer more targeted comments but one thing to keep in mind is you want to use some sort of phasing strategy (an example here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472414/) to ensure that not all amplicons have the same base at a particular cycle.

Each cluster generates reads (one read if single-end sequencing, two (from two ends of the fragment) if paired-end sequencing). Illumina sequencing works best when bases A/C/G/T are evenly distributed across the clusters (since the software is locating/keeping track of clusters).
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Old 04-14-2016, 07:14 PM   #3
nucacidhunter
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If you have not included phasing bases and amplicon sequences are low diversity as GenoMax has mentioned, you will have to spike-in 20% PhiX and cluster at lower density (600-700). This should give over 10M demultiplexed reads from your amplicons which would give 120-130x average coverage (10M/87,000). You can do one more run if the read numbers are very uneven or not sufficient for your analysis.
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Old 04-15-2016, 09:08 AM   #4
thermophile
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You will not likely have the same number of amplicons from each sample (sorry, pooling exactly accurately is impossible. If my amplicons have less than an order of magnitude difference in sequences, I consider it a win). I have seen people have better pooling success using a normalization plate but I doubt that would be cost effective for you given your sample numbers.

Second, I'd stay away from the 2x300 kit. Even through your amplicons are 300bp, I bet you'll have better results using the 2x250 kit.

Are you only using one target primer set to generate your amplicons? (as in you're only doing one region of 18s or only doing co1) Or are you running many amplicons from different regions? If it's only one, you're going to have to spike in a lot of phiX (at least 20%) because you'll be sequencing your target primers first which are identical.
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Old 04-15-2016, 01:38 PM   #5
sme.bug
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Thank you GenoMax and nucacidhunter! Thermophile, my amplicons are all from the same region but each sample will have a different 8 bp identification tag added to the start of the primers. Would that give enough variety?

Why would I expect better results from the 2x250 kit?
Thanks!
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Old 05-07-2016, 11:03 AM   #6
LOH
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same question to thermophile,
why will it be better results by using 2x250 V2 reagent?
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Old 05-09-2016, 09:56 AM   #7
thermophile
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the quality of the reads decreases faster for the v3 kits than v2. Many people are seeing the same thing, check out the mothur blog for details on their experience which is similar to my few tries with v3.

http://blog.mothur.org/2014/09/11/Wh...stance-matrix/
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Old 05-09-2016, 12:43 PM   #8
barrmur
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Quote:
Originally Posted by thermophile View Post
the quality of the reads decreases faster for the v3 kits than v2. Many people are seeing the same thing, check out the mothur blog for details on their experience which is similar to my few tries with v3.

http://blog.mothur.org/2014/09/11/Wh...stance-matrix/
Fully agree, just remember to account for the lower amount of reads when using the v2 500 cycle kit as opposed to the v3 600 cycle kit.
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Old 05-09-2016, 03:04 PM   #9
fanli
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Like other posters said, do the 2x250 V2 and then stitch the reads together afterwards. Stay away from the 2x300 kit until Illumina fixes it...

Also, it's worth noting that there is instrument to instrument variability that may affect how much PhiX you have to spike in to generate sufficient diversity. We got a good one - last run of 16S had 91.9% Q30 at 1039 K/mm2 with no PhiX.
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Old 05-09-2016, 08:06 PM   #10
nucacidhunter
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Quote:
Originally Posted by fanli View Post
Like other posters said, do the 2x250 V2 and then stitch the reads together afterwards. Stay away from the 2x300 kit until Illumina fixes it...

Also, it's worth noting that there is instrument to instrument variability that may affect how much PhiX you have to spike in to generate sufficient diversity. We got a good one - last run of 16S had 91.9% Q30 at 1039 K/mm2 with no PhiX.
I wonder if you use custom sequencing primer and also if you included some sort of diversity spacer to stagger the reads. Output sounds very good almost approaching a shotgun library.
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Old 05-10-2016, 08:06 AM   #11
fanli
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Nope, these are the "old" EMP primers. We probably should still spike PhiX to buffer against run issues...but sometimes it gets late and the bench folks want to go home too
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