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Old 02-25-2016, 12:28 PM   #1
urchin
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Smile 16s primer design for Illumina platform

I am another newbie trying to get through a first project in NGS.

We are trying to amplify universal prokaryotic sequences for 16s rRNA gene (V3-V4 region) from benthic marine critters - mostly from microbiome extractions using materials from invertebrates and some algae. I was told that using the Fadrosh et al. 2014 dual-indexing method would improve our results a lot when we do PE250. Hopefully that is true - yes? (No?)

Also, right now all the current primer sets are going through continued evaluation and most seem to be found lacking in one way or another. Does anyone have a strong opinion on choices based on actual experience with getting good results? Has anyone tried the primers discussed in Takahashi et al. 2014, Pro341F/Pro805R? Did they work as well as claimed?

Thanks for any help pointing me in the right direction!
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Old 05-25-2016, 07:14 AM   #2
jtotheulie
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Hi,

Do you have an update on this project? Did you test Fadrosh et al. 2014 dual-indexing method?

thanks
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Old 05-25-2016, 03:03 PM   #3
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Hello, No I did not. I am using the Kozich method. I was able to get in touch with a friend's colleague who is doing this type of work already and he informed me that the problems with libraries with low diversity getting good reads with Illumina Miseq for PE250 have been resolved with new chemistry. He said the additional modifications really weren't necessary anymore.

Anyone have another opinion about that??
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Old 05-26-2016, 04:16 AM   #4
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Quote:
Originally Posted by urchin View Post
We are trying to amplify universal prokaryotic sequences for 16s rRNA gene (V3-V4 region) from benthic marine critters - mostly from microbiome extractions using materials from invertebrates and some algae. I was told that using the Fadrosh et al. 2014 dual-indexing method would improve our results a lot when we do PE250. Hopefully that is true - yes? (No?)

Thanks for any help pointing me in the right direction!
For optimum quality 16S libraries has to be spiked in with high diversity libraries and clustered in lower density regardless of improved chemistry. Adding heterogeneity spacer will enable increasing cluster numbers therefore maximising sequencing yield without adverse effect on quality. The issues with the method are:

1- Adding inline index decreases useful read length. This is OK for short target regions but unsuitable for longer targets such as V1-V3.
2- There are some possible issues with reproducibility using different spacers that have not been addressed by the authors (they have not used a known control community with all of their primers to see if they get the same results).
3- Long primers are less efficient and more biased in amplification.
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Old 05-26-2016, 11:42 AM   #5
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The Illumina sequencing site we are sending our library to will use Phix to spike the library. I'm guessing at around 30%?? Is that adequate to deal with this?

I'm not clear if you are really trying to recommend the spacers or not with your additional points it seems they could be a problem for us.

We are planning on amplifying V3-V4 using 341f and 785r, not quite as long as V1-V3 so would the inline index present a problem for our target length getting read adequately?
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Old 05-26-2016, 04:23 PM   #6
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Quote:
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The Illumina sequencing site we are sending our library to will use Phix to spike the library. I'm guessing at around 30%?? Is that adequate to deal with this?

I'm not clear if you are really trying to recommend the spacers or not with your additional points it seems they could be a problem for us.

We are planning on amplifying V3-V4 using 341f and 785r, not quite as long as V1-V3 so would the inline index present a problem for our target length getting read adequately?
20% PhiX with cluster density below 600 would be fine.

Generally spacers are good and one can increase cluster density and reduce PhiX spike in (more reads). The issue is that one have to validate their design to make sure that spacer does not cause any biases. One way to do this would be using control mock community with known species and amplifying target V region followed by sequencing and analysis. If there is no bias all primers should give very similar quantitative and qualitative results. The paper has not mention the validation.
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Old 06-01-2016, 05:16 AM   #7
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Has anyone modified the 16s illumina protocol for other amplicons. I want to use it for 16s, its, and 18s. If we can do this then we can use the same sequencing primers and sequence them in the same run. Avoiding the conserved sequencing issue that illumina has and the confusion of the EMB protocol that uses different sequencing primers between the 16 and 18s.

Any thoughts?
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Old 06-02-2016, 07:05 AM   #8
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I've modified Kozich primers for ITS and family specific portions of 16s. Doing this requires spiking in a sequencing primer for each target (the sequencing primer is the pcr primer plus a pad). This works well. If you're running lots of samples with a particular target, I'd recommend this method since you don't waste sequencing cycles on your pcr primers.

But for those cases where you aren't running lots of samples on a target, I'm also doing a 2 step target agnostic amplicon design based on http://bmcgenomics.biomedcentral.com...471-2164-15-63
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Old 06-20-2016, 10:32 AM   #9
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Default phasing amplicon approach - alternate spacers?

Quote:
Originally Posted by nucacidhunter View Post
20% PhiX with cluster density below 600 would be fine.

Generally spacers are good and one can increase cluster density and reduce PhiX spike in (more reads). The issue is that one have to validate their design to make sure that spacer does not cause any biases. One way to do this would be using control mock community with known species and amplifying target V region followed by sequencing and analysis. If there is no bias all primers should give very similar quantitative and qualitative results. The paper has not mention the validation.
The Fadrosh work has now been followed up with a modification on the spacers by Wu et al. 2015. Do you think that the control tests they did are adequate? I am now considering using this method for our primer spacers instead of just depending on the chemistry at Illumina being good enough for the lack of diversity. This method will not cause us to lose so much of our sequencing length, as well.

By the way, does anyone think there is a problem with using PE300 instead of PE250 with this method????
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Old 06-21-2016, 04:24 AM   #10
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Could you give more details for Wu et al 2015 to clarify the paper you are referring.
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Old 06-21-2016, 08:29 AM   #11
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Quote:
Originally Posted by nucacidhunter View Post
Could you give more details for Wu et al 2015 to clarify the paper you are referring.
Wu et al. BMC Microbiology (2015) 15:125
DOI 10.1186/s12866-015-0450-4

Phasing amplicon sequencing on Illumina Miseq for
robust environmental microbial community analysis
Liyou Wu1, Chongqing Wen1,3, Yujia Qin1, Huaqun Yin1,4,5, Qichao Tu1, Joy D. Van Nostrand1, Tong Yuan1,
Menting Yuan1, Ye Deng1,7 and Jizhong Zhou1,2,6*

They have spacers that always are 7 bases long, by altering the spacer insert length on 5' versus 3' primers. They did include various control tests, including against a mock community.

Here is a quote from the abstract: "Our results first indicated that the PAS method substantially ameliorated the problem of
unbalanced base composition. Second, the PAS method substantially improved the sequence read base quality
(an average of 10 % higher of bases above Q30). Third, the PAS method effectively increased raw sequence
throughput (~15 % more raw reads). In addition, the PAS method significantly increased effective reads (947 %)
and the effective read sequence length (1696 more bases) after quality trim at Q30 with window 5. In addition,
the PAS method reduced half of the sequencing errors (0.541.1 % less). Finally, two-step PCR amplification of
the PAS method effectively ameliorated the amplification biases introduced by the long barcoded PCR primers."
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Old 06-21-2016, 12:57 PM   #12
urchin
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Default Link to Wu et al 1015

http://bmcmicrobiol.biomedcentral.co...866-015-0450-4
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Old 06-21-2016, 01:49 PM   #13
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Quote:
Originally Posted by urchin View Post

By the way, does anyone think there is a problem with using PE300 instead of PE250 with this method????
The 600 cycle v3 kits were showing really poor base qualities toward the end of reads. I am not sure if Illumina has resolved this yet.
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Old 06-21-2016, 11:23 PM   #14
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@urchin

Wu's work seems has addressed the amplicon sequencing issues on Illumina platforms adequately. I would suggest a small trial if you are amplifying other regions or modifying their protocol with one control sample.
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Old 06-22-2016, 09:43 AM   #15
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Thanks for the information, I thought it looked like they had done a relatively thorough job, but I am new to this area.

I wanted to do V3V4 but now am very concerned that I won't get enough sequence length to overlap for alignment if I have to use PE250 to get decent reads. Above "Kerplunk412" indicated the PE300 is not giving good quality reads at the ends, so I don't see how that would help me get more useable data. Does anyone have possible suggestions or knowledge about what specific sequence length I could get with PE300?
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Old 06-22-2016, 02:58 PM   #16
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My caution is because they have reported results for 3 primer pairs.

For your region of interest 300PE is essential even though current 600 cycle reagents gives inconsistent results. Overal Q scores for the 3' end of reads is low but a subset of reads stil have good quality. Phasing reads increases yield by enabling higher cluster density and lower Phix spike-in (at least 1.5x) and therefore increases reads with good quality 3' end.

I think Wu's method has set good benchmark if their reported lack of biase can be shown in other experiments as well. Addition of 7 base spacer would have minimal effect on the number of assembled paired reads.
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Old 06-23-2016, 07:05 AM   #17
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their benchmarks are for just v4 (~275bp) on 500 cycle v2 chemistry. You're going to have to do your own experiments to see if that can be extrapolated to v3-4 (~550bp) on v3 chemistry (which seems to cause everyone issues for amplicon sequencing).

My opinion, staggered primers aren't worth the effort. Two step PCR introduces errors on it's own. I'm happy multiplexing a couple hundred samples per run which gives me plenty of sequences/sample without trying to max out the cluster density.
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Old 06-23-2016, 03:07 PM   #18
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@thermophile

Staggered primers can increase output 1.5-2x resulting in decreased sequencing cost.

To my knowledge no current community profiling method has been shown to be biase free or ever been tested except Wu's work.

I wonder what number of reads per sample you consider adequate.
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Old 06-25-2016, 06:57 AM   #19
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Miseq data, i rarify to 10k seqs for analysis. Unless you are searching for very rare members of the community, even that is likely overkill. A decade ago we were using 100 clones/sample, then 1000-2500 seqs/sample for 454 data and finding similar patterns.

People that ask me for more than 10k seq/sample and that aren't searching for rares, i urge to analyze more samples instead. Either more time points or more technical replicates-extract more than one 1/4 g per sample rather than just sequence the same 1/4g over and over.

This advice is especially true for anyone who's bioinformatic processing includes a step that removes all sequences below a certain % of the community (which is a pretty common practice). If you're only interested in the most abundant organisms (anything over 1% or .5% of the community is abundant when talking bacterial communities), why spend money on sequencing the rares?
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Old 06-25-2016, 08:13 AM   #20
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I have seen absolutely terrible quality from 2x300bp Illumina kits in amplicon sequencing, to the point that the data was completely unusable with (IIRC) >15% error rate. I have not seen any Illumina 2x300bp amplicon data of good quality. That doesn't mean it doesn't exist, because I don't look at all of our data, but I have not seen it. I have, however, seen good quality 2x250bp amplicon data.

At JGI we do use staggered primers, which I always thought was an excellent decision because it greatly increases the color diversity during sequencing. I was unaware that they might increase bias; could you explain that?

As for rarifying to 10k sequences (I would call it subsampling), that will clearly reduce your statistical power. To achieve cost-savings... how do you even multiplex to the point that you get 10k reads per sample? And removing anything under 1%... yes, certainly, that makes deep sequencing unnecessary (even if you define "deep" as a few thousand reads ) but whether or not it is common practice, that seems like an incredibly bad idea to me. I think that if you have enough reads to form a cluster that is statistically distinct from clusters from coincidental matches of high-error or incorrectly-merged reads, and therefore indicates an active species, you can gain knowledge from it. As you noted, amplification causes bias. Potentially, a species present at 0.9% in your amplified sequencing could be 20% of your actual community that was outcompeted at the amplification stage, right? In which case, it would in fact be very important to compare at different time-points, etc. Or it could actually represent 0.9% of the community that performs a crucial role of, say, mobilizing a specific element like iron rather than doing the bulk work of eating a specific carbon-containing molecule. Which one is interesting depends on your research, of course, but a community is a community because it is diverse, and presumably the low-abundance members of the community are essential to the function of the community, or else they would not exist.
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