Protist diversity—454 or Illumina？

[lingnanwu]

Hi,I'm a newbie in NGS.
Our groups are going to study the diversity of protist community,the target region—18s rRNA V9 region，length is about 150bp.I think Illumina is enough,but in many articles they all use 454.So I want to know if some one knows why they all use 454,which sequencing technology should I choice?Thank you.

[nickloman]

If the region is only 150bp long, I'd be inclined to use Illumina (MiSeq would be ideal as it supports 150bp reads) because it will be much more cost-effective than 454. The 454 is a better option if you need the longer reads.

[rglover]

ah, a double posting - this post is out in another area of the forum too. I'd be inclined to use 454, despite the cost, because of the error rate in illumina increasing the number of 'species' artificially...good data is worth the cost?

[nickloman]

It's not like 454 doesn't do this Rachel - hence the need for expensive denoising algorithms like AmpliconNoise. The dominant type of errors is different though; 454 suffers more from indels associated with homopolymers, Illumina more from substitution errors. But you need to clean up your data from either platform if you don't want the false OTU inflation problem. It's worth mentioning that Illumina doesn't do great with low diversity amplicons and you need to factor that in somehow, e.g. having poly-Ns in your primer.

[nickloman]

This is a useful reference about Illumina 16S sequencing:

http://www.nature.com/ismej/journal/v6/n8/full/ismej20128a.html

[rglover]

454 suffers more from indels associated with homopolymers, Illumina more from substitution errors

tbh that's one of the main reasons we use 454. A lot of the amplicon studies we do are protein-coding amplicons (although they're quite long compared to the OP's) and so it's easier to spot and correct frameshift-inducing homopolymer errors than substitutions which could increase the diversity of OTUs in the sample. Did some 600bp COI invertebrate DNA barcode amplicons a while back - AT rich mitochondrial sequences with 'normal' coding homopolymers >6bp...

[nickloman]

tbh that's one of the main reasons we use 454. A lot of the amplicon studies we do are protein-coding amplicons (although they're quite long compared to the OP's) and so it's easier to spot and correct frameshift-inducing homopolymer errors than substitutions which could increase the diversity of OTUs in the sample. Did some 600bp COI invertebrate DNA barcode amplicons a while back - AT rich mitochondrial sequences with 'normal' coding homopolymers >6bp...

True, if you are looking at protein coding stuff then you could just toss the indels to make life easier (obviously means you can't detect real indels though). The 454 does also have substitution errors though, hence some kind of clustering step is needed. If you have a 1% error rate, say, then clustering at 3% is somewhat reasonable to get rid of most sequence errors (made-up numbers).

[HMorrison]

v9 on Illumina vs. GSFLX

rRNA amplicon sequencing is 90% of what we do; we used 454 because that's what was available to do 'long' reads back in 2005. Recall that the original Solexa reads were 35 nt. We're shifting 18s v9 sequencing and 16s v6 and v4-v5 to Illlumina now that reads are longer.

Hilary

[lingnanwu]

rRNA amplicon sequencing is 90% of what we do; we used 454 because that's what was available to do 'long' reads back in 2005. Recall that the original Solexa reads were 35 nt. We're shifting 18s v9 sequencing and 16s v6 and v4-v5 to Illlumina now that reads are longer.

Hilary

Thank you,Hilary.Can you tell me the primer you use to amplify the V9 region of 18s?We want to use 1391f/EUKB or 1391f/1510r.

[HMorrison]

v9 primers

Thank you,Hilary.Can you tell me the primer you use to amplify the V9 region of 18s?We want to use 1391f/EUKB or 1391f/1510r.

We have used

1510R, CCTTCYGCAGGTTCACCTAC

with a combination of
1380F CCCTGCCHTTTGTACACAC
1389F TTGTACACACCGCCC

Citation is Stoeck, T., Behnke, A., Christen, R., Amaral-Zettler, L. A., Rodriguez-Mora, M., Chistoserdov, A., Orsi, W. & Edgcomb, V. 2009, Massively parallel tag sequencing reveals the complexity of anaerobic marine protistan communities, BMC Biology, vol. 7, no. 1, p. 72.