MiSeq 16S - a couple of questions

cliffbeall · 10-30-2014, 12:04 PM

We have just started to sequence 16S on the MiSeq and there were a couple of points that I wanted to get feedback on.

One is that we followed the Illumina publication "16S Metagenomic Library Prep Guide", substituting V1V3 primers for the V3V4. That says if you use the latest software you can get by with 5% PhiX but after we submitted the library our core informed us they would have to put in 50% PhiX, and that we really should have used an alternative protocol from Ravel's lab with variable length spacers.

The other thing is that when we do amplifications with universal 16S primers we get a band of expected size with a smear above it. We're fairly sure these are heteroduplexes. When we have sequenced preps like this on 454 there is no evidence of longer fragments or non-16S amplicons. There are multiple publications on 16S heteroduplexes, it's a recognized phenomenon. Nonetheless, our sequencing core wants to size select the library on a Pippen prep, which I don't want to do because I really think its unnecessary and may introduce bias. They are telling me if I don't let them size select they won't guarantee the results, if they fail (for any reason) we are on the hook.

I would appreciate any input:
--does that Illumina protocol not work as advertised?
--Are some cores still using the old software for whatever reason?
--Do you regularly size select 16S preps for MiSeq?
--Have you compared size selected to non-sized to look for bias?

nucacidhunter · 10-31-2014, 03:24 AM

10% Phix spike in, 80% of normal library (high sequence diversity) pM input for clustering should give good quality reads. 50% Phix was for old software and I do not see any reason for not updating to new version of software.

I agree with you, size selection is unnecessary (I have not heard anyone doing it) and clustering bias towards smaller amplicons would take care of large ones if they are present. If it is their policy you may choose other provider. Generally sequencing centres guarantee for in-house and client prepared libraries are different.

MU Core · 10-31-2014, 03:27 AM

If running the library on a MiSeq you will not need to use 50% PhiX. Our facility uses 10% and obtain good results. The 50% PhiX spike-in is the recommendation for low diversity libraries ran on a HiSeq. Even this has changed, however, with the recently released (August) HCS version which allows the HiSeq to better handle low diversity libraries in the same manner as a MiSeq.

dfhdfh · 11-04-2014, 01:17 AM

I'm also planning some 16 S sequencing on a MiSeq and read that the Illumina protocol isn't the best for doing that. What do you guys recommend?

nucacidhunter · 11-04-2014, 02:41 AM

Quote:

I'm also planning some 16 S sequencing on a MiSeq and read that the Illumina protocol isn't the best for doing that.

Do you have a reference?

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10-30-2014, 12:04 PM	#1
cliffbeall Senior Member Location: Ohio Join Date: Jan 2010 Posts: 144	MiSeq 16S - a couple of questions We have just started to sequence 16S on the MiSeq and there were a couple of points that I wanted to get feedback on. One is that we followed the Illumina publication "16S Metagenomic Library Prep Guide", substituting V1V3 primers for the V3V4. That says if you use the latest software you can get by with 5% PhiX but after we submitted the library our core informed us they would have to put in 50% PhiX, and that we really should have used an alternative protocol from Ravel's lab with variable length spacers. The other thing is that when we do amplifications with universal 16S primers we get a band of expected size with a smear above it. We're fairly sure these are heteroduplexes. When we have sequenced preps like this on 454 there is no evidence of longer fragments or non-16S amplicons. There are multiple publications on 16S heteroduplexes, it's a recognized phenomenon. Nonetheless, our sequencing core wants to size select the library on a Pippen prep, which I don't want to do because I really think its unnecessary and may introduce bias. They are telling me if I don't let them size select they won't guarantee the results, if they fail (for any reason) we are on the hook. I would appreciate any input: --does that Illumina protocol not work as advertised? --Are some cores still using the old software for whatever reason? --Do you regularly size select 16S preps for MiSeq? --Have you compared size selected to non-sized to look for bias?

10-31-2014, 03:24 AM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,235	10% Phix spike in, 80% of normal library (high sequence diversity) pM input for clustering should give good quality reads. 50% Phix was for old software and I do not see any reason for not updating to new version of software. I agree with you, size selection is unnecessary (I have not heard anyone doing it) and clustering bias towards smaller amplicons would take care of large ones if they are present. If it is their policy you may choose other provider. Generally sequencing centres guarantee for in-house and client prepared libraries are different.

10-31-2014, 03:27 AM	#3
MU Core Member Location: Columbia, Missouri Join Date: Apr 2008 Posts: 57	If running the library on a MiSeq you will not need to use 50% PhiX. Our facility uses 10% and obtain good results. The 50% PhiX spike-in is the recommendation for low diversity libraries ran on a HiSeq. Even this has changed, however, with the recently released (August) HCS version which allows the HiSeq to better handle low diversity libraries in the same manner as a MiSeq.

11-04-2014, 01:17 AM	#4
dfhdfh Member Location: Germany Join Date: Jan 2013 Posts: 46	I'm also planning some 16 S sequencing on a MiSeq and read that the Illumina protocol isn't the best for doing that. What do you guys recommend?