Things about 16S 515F-806R

[jt wang]

Dear all,

    I have been analyzing on a dataset of 40 soil samples that genearted in a Illumina Miseq run. Primer set 515F-806R targetting V4 region of 16S rDNA was employed. This primer set was supposed to available for both bacteria and archaea. However, as you see in the following table, the archaeal seqs number detected in each sample was so poor. Is it normal for forest soils? Or there may be PCR bias? 

sample ID seqs per sample	S4 125788	S5 375844	S14 127427	S15 123203	S34 151404	S35 79758
Unclassified;Other;Other	20	5	133	19	85	2
k__Archaea;p__Crenarchaeota;c__MBGA	28	79	153	49	44	3
k__Archaea;p__Crenarchaeota;c__MCG	0	0	0	0	0	152
k__Archaea;p__Crenarchaeota;c__Thaumarchaeota	1	10	2	2	3	32
k__Archaea;p__Euryarchaeota;Other	5	41	1	0	2	1
k__Archaea;p__Euryarchaeota;c__Methanobacteria	0	3	0	0	0	13
k__Archaea;p__Euryarchaeota;c__Methanomicrobia	0	1	0	0	0	63
k__Archaea;p__Euryarchaeota;c__Thermoplasmata	0	17	0	0	0	0
k__Archaea;p__Euryarchaeota;c__[Parvarchaea]	2	2	10	0	0	23
total Arc seqs	36	153	166	51	49	287

PS: Sequence quality contral was performed by the sequencer. I use gg_12_10_otus_qiime_linked database for assigning(97%). Singletons were removed from the otu table. Barcoded primers were synthesized according to JG Caporaso,2012(http://www.nature.com/ismej/journal/v6/n8/full/ismej20128a.html). 

[Tony Walters]

Hello,

Here's a paper about some previous observations regarding archaeal distributions in soil (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105767/) (relates to carbon/nitrogen ratio).

For the primer set, the reverse primer should have perfect matches for almost all archaea, the forward primer 515f has a perfect match for most Euryarchaeota, and a single mismatch towards the 5' end (C-A mismatch) for Crenarchaeota.  The primer sequence looks like this: GTGCCAGCMGCCGCGGTAA, and target Crenarchaeota sequences look like this: GTGTCAGCCGCCGCGGTAA, which should have minimal impact being near the 5' end of the primer.

-Tony

--
 
---
You received this message because you are subscribed to the Google Groups "Qiime Forum" group.
To unsubscribe from this group and stop receiving emails from it, send an email to qiime-forum...@googlegroups.com.
For more options, visit https://groups.google.com/groups/opt_out.
 
 

[jt wang]

Thanks, Tony. I did read the paper you mentioned, and noticed the lower relative abuntance of archaea in humid conifer forest soils like MEW138 and MEW139, which were similar to mine. However, I was wondering if the Illumina adaptors that linked to the reverse primer 806R had any PCR bias effects? Thank you. 

在 2013年5月25日星期六UTC+8下午12时22分43秒，TonyWalters写道：

[Tony Walters]

I don't know of a particular bias due to the Illumina adapter-you might compare the adapter sequence to the sequence downstream from the 806r primer site to a handful of archaea/bacterial sequences to see if there is much of a match between the adapter sequences and the 16S gene (although from blasting some of the Illumina adapters, they seem to be very poor at matching 16S data, so my guess is that there would be minimal binding of the adapter across taxa, and even if there are some matches, at this 5' end of the primer construct it probably has minimal effect).

[jt wang]

Actually, after the communication with the sequencer, I have decided to reduce the cycles from 30 to 20 to see if there would be any improvement for the same sample. I appreciate your advice, Tony. Many Thanks.

在 2013年5月25日星期六UTC+8下午2时20分27秒，TonyWalters写道：