sequencing 16s rRNA

[Nullie]

Is it possible to sequence the 16s rRNA of bacteria using RNA-SEQ?

If not, how can I sequence it and use that to check for different bacteria ditribution in my sample?

[SNPsaurus]

There are specialized protocols for 16S sequencing, since you are just sequencing a region rather than a transcriptome. See this Illumina white paper for an example: http://www.illumina.com/documents/pr..._miseq_16S.pdf

[vacuna]

16s rRNA

i'm too looking for a method for rRNA sequancing of 16S rRNA, but not DNA : RNA

[pedrolcalves]

Can anyone tell how much time a ~300 bp sequence would take to sequence? Manufacturer page says around 39 hours but it refers to 2x350 bp. What does the "2x" mean?

[nucacidhunter]

If it is an Illumina sequencer 2x means paired end sequencing. Illumina's longest read would be 2x300 meaning 300 cycles for each of Read1 and Read2 (600 base in total). Maybe you meant 2x150.

[pedrolcalves]

Quoting from the illumina site: "read length: 2 × 250 bp; total time:~39 hrs"
Does it mean paired-end read?
In this case, is it suitable to do a single read and would it take less time? Would it be close to half?
Thank you for your reply

[Brian Bushnell]

Yes, the time is mostly determined by the number of cycles, so 2x250 = 500 cycles; 300bp should take around (300/500)*39 hours plus some overhead.

Edit: (300/500)*39 = 23.4

[pmiguel]

Quoting from the illumina site: "read length: 2 × 250 bp; total time:~39 hrs"
Does it mean paired-end read?

Yes.

In this case, is it suitable to do a single read and would it take less time? Would it be close to half?
Thank you for your reply

It would be a single 250 base read for each cluster.
I wouldn't recommend it unless you have money to burn. Higher quality sequence and less cost if you can create 250 bp amplicons and sequence them using 2x150 (300 cycle) cassette. That takes right around 24hours, if memory serves.

--
Phillip

[Brian Bushnell]

As Phillip says, 2x150 has advantages over 1x300; you can subsequently merge the paired reads to create single sequences for analysis. But the length will be shorter, since you need to allow sufficient overlap to be unambiguous.

Note that Illumina's 300bp kits have been very troublesome for a while and Illumina amplicon sequencing always has a much higher error rate than shotgun sequencing. As such, I would not really recommend Illumina 300bp sequencing for anything at present. I think that currently, if you have access to a MiSeq, for 16S, 2x250bp with at least 100bp overlap (so, 400bp insert) is optimal, though you could stretch that to a 450bp insert. It will certainly give you longer and higher-quality sequences than 1x300bp, assuming you use a good read-merging program.

I don't personally deal much with 16S sub-regions, but I work with analysts that do, and they tend to dislike very short 16S sections (<300bp) because it makes it hard for them to be certain about what organism they are working with when they try to determine whether the sample is correct or contaminated.

[pmiguel]

Yes, I agree with Brian, although when last I inquired Illumina was trying to address issues with the 600 cycle kit (and 500 cycle, at least for a while). Not sure where they are with that.

One option that would give you speed initially would be to do the 500 cycle run, 2x250 bases, but process your data after the first 3 of the 4 reads (read1 index1 index2 read2) are collected. That should happen in less than 20 hours.

Then you can refine your results half a day later, if desired, using the reverse reads.

I think you would have to do the data processing off-instrument to be able to do this though.

--
Phillip

[thermophile]

What are you trying to identify? Paired end will give you much better quality as long as you have near complete overlap. Since you are looking for very small differences between sequences, you need that very high overlap.

If you are trying to very quickly identify certain organisms, it's possible/likely that 16s isn't the appropriate target