Question: Number Of Samples Per Lane For Multiplexed Rna-Seq
3
gravatar for Dave Bridges
8.7 years ago by
Dave Bridges1.3k
Ann Arbor, MI
Dave Bridges1.3k wrote:

We are trying to decide how many multiplexed samples to use for a RNA-seq experiment. If we expect ~50M usable reads per lane and presume a 50K transcriptome to get ~1000 reads per gene, then 5 multiplexed samples would give 200 reads per "average" transcript. Is that level of coverage reasonable for differential expression analysis, or should we aim for higher coverage?

ADD COMMENTlink written 8.7 years ago by Dave Bridges1.3k

Just remember that genes have differing levels of expression; you will get a large proportion of your reads mapping to the most highly expressed genes.

ADD REPLYlink written 8.7 years ago by Gww2.7k
2
gravatar for Istvan Albert
8.7 years ago by
Istvan Albert ♦♦ 84k
University Park, USA
Istvan Albert ♦♦ 84k wrote:

I would recommend reading some of the relevant publications, a good start would be:

RNA-Seq: a revolutionary tool for transcriptomics

ADD COMMENTlink written 8.7 years ago by Istvan Albert ♦♦ 84k
2
gravatar for Travis
8.7 years ago by
Travis2.8k
USA
Travis2.8k wrote:

Reads required will be dependent on your desired application/analysis also.

Some numbers recommended by Illumina for human transcriptome sequencing:

3´-SAGE:            1-5 M reads
WT-GE:              5-10 M reads
Alt splicing:       10-50 M reads
Novel Transcripts:  >50 M reads
ADD COMMENTlink written 8.7 years ago by Travis2.8k

is WT-GE normal gene expression for differential comparason?

ADD REPLYlink written 8.7 years ago by Dave Bridges1.3k

Got a link for those numbers?

ADD REPLYlink written 8.7 years ago by Aaron Statham1.1k

Aaron, the numbers came directly from a user-group presentation so no link unfortunately. Dave, yes so far as I am aware.

ADD REPLYlink written 8.7 years ago by Travis2.8k
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