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Old 06-04-2015, 02:47 AM   #1
psax
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Default Nextseq 500 amplicon length considerations?

Hi guys,
I'm planning a bacterial 16s rRNA amplicon sequencing project using Nextseq 500 with 300-cycles reagents. Now I'm trying to figure out how to choose an amplicon size and what the best run type is (single or paired end? If the latter, should the two paired end reads partially overlap?). Are there any basic "rules" or reliable primer sets available? Any suggestions/references would be appreciated.
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Old 06-04-2015, 10:05 AM   #2
Brian Bushnell
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It's best to run paired-end 2x150 with overlapping reads (design for at least 30bp overlap). This will greatly limit your set of available regions; if at all possible, I'd suggest 2x250 or 2x300 on a MiSeq.
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Old 06-06-2015, 06:13 PM   #3
psax
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Quote:
Originally Posted by Brian Bushnell View Post
It's best to run paired-end 2x150 with overlapping reads (design for at least 30bp overlap). This will greatly limit your set of available regions; if at all possible, I'd suggest 2x250 or 2x300 on a MiSeq.
Thank you Brian. I will start from there
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Old 06-07-2015, 03:44 PM   #4
bilyl
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Quote:
Originally Posted by psax View Post
Hi guys,
I'm planning a bacterial 16s rRNA amplicon sequencing project using Nextseq 500 with 300-cycles reagents. Now I'm trying to figure out how to choose an amplicon size and what the best run type is (single or paired end? If the latter, should the two paired end reads partially overlap?). Are there any basic "rules" or reliable primer sets available? Any suggestions/references would be appreciated.
Commerically available V4 kits play well with 2x150. You could do a V1-V2 amplicon but you'll need longer reads to do that.

As for the NextSeq, you'll have a really bad time unless you can base stagger your amplicons when you multiplex. Even with a healthy dose of PhiX you'll get poor quality as compared to the MiSeq. I suggest you read up on the primers used and homebrew your own kit.
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Old 06-07-2015, 04:24 PM   #5
Brian Bushnell
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Furthermore, NextSeq has way more capacity than is needed for a 16s study, and currently has major problems with multiplexing; and the 4 "lanes" are not separated. So even if it supported longer reads, I would not recommend it for this purpose.
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Old 06-14-2015, 07:49 PM   #6
psax
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Quote:
Originally Posted by bilyl View Post
Commerically available V4 kits play well with 2x150. You could do a V1-V2 amplicon but you'll need longer reads to do that.

As for the NextSeq, you'll have a really bad time unless you can base stagger your amplicons when you multiplex. Even with a healthy dose of PhiX you'll get poor quality as compared to the MiSeq. I suggest you read up on the primers used and homebrew your own kit.
Thank you for your advices. With base stagger, do I still have to add 30-50% PhiX?
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Old 06-15-2015, 01:03 PM   #7
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I think you'll find more than a few posts recommending that you NOT use NextSeq for amplicon sequencing, even with PhiX. It simply doesn't work as well as MiSeq.
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