Question: MiSeq or HiSeq for metatranscriptome sequencing
0
gravatar for mariaroviscomonteiro
9 months ago by
mariaroviscomonteiro0 wrote:

Hello,

I have a question. I have 6 samples in which I want to do metatranscriptomic sequencing, but my question now is whether I should use MiSeq or HiSeq technologies. I know HiSeq would give me much more reads depth but I am concerned about reads length as well. I am working with Antarctic samples so the microbial community is quite simple, however the organisms I might encounter are also not very well described, so longer reads would possibly help me further up with the assembly.

Any thoughts or experience about this?

Thank you!

sequencing rna-seq assembly • 520 views
ADD COMMENTlink modified 9 weeks ago by Carambakaracho620 • written 9 months ago by mariaroviscomonteiro0

Is your goal the metagenome assembly or to asses the functional activity of the microbial community?

ADD REPLYlink written 9 months ago by Carambakaracho620

That's not metagenome assembly, the OP is interested in metatranscriptomic assembly

ADD REPLYlink written 9 weeks ago by Vijay Lakhujani3.4k

OP is interested in metatranscriptomic assembly

which means he would like to

asses the functional activity of the microbial community

Admitted, this wasn't phrased very clear. Anyway, OP also remained very silent for a few months

ADD REPLYlink modified 9 weeks ago • written 9 weeks ago by Carambakaracho620

Hi, any updates on that? I am having the same question here

ADD REPLYlink written 9 weeks ago by Leonardo30

In case your microbiome has a very low complexity or you're interested in the most abundant species, and you expect MiSeq coverage suffcient, the longer reads will help a little bit to improve the assembly. It's still short reads, so don't expect miracles. In most other cases the increased coverage will be more advantageous.

Keep in mind that 16S amplicon seq is a whole different story.

ADD REPLYlink written 9 weeks ago by Carambakaracho620

When you consider transcriptome for study, then read length of 2x150 bp is enough (reference) to get good assembly unlike genome analysis where you can obtain good results with increased read length. As you are working with metagenome you can focus more on considering more read depth so as to improve the assembly result. With more number of samples in hand, you can go for Hiseq with 2x250 bp chemistry which can give you comparable result like Miseq 2x300bp chemistry and better sequencing coverage.

ADD REPLYlink modified 9 weeks ago • written 9 weeks ago by toralmanvar720

hi @toralmanvar though I agree in principle, in my experience, I had little improvement with longer Illumina read lengths in both WGS and WMS.

For RNAseq, the paper you cited focuses on eukaryote transcriptome assembly. In prokaryotes, which make up most of what most people refer to as a microbiome, the genome is much denser. There's no intragenic region between many genes and the short existing intragenic regions are often transcribed as well. In terms of total sequence coverage, the metatranscriptomes I analyzed were not much different from their respective metagenomes (I'm not speaking of the coverage profile, I mean something like a binary decision as in has or has no coverage).

ADD REPLYlink modified 9 weeks ago • written 9 weeks ago by Carambakaracho620
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