Paired-end & shotgun reads

[nickloman]

Hi there

Can anyone help me with this conundrum? Roche state in a flyer that you should be able to sequence E. coli (~4mb) using only the 8kb paired-end protocol to 20x coverage on a 1/4 plate, suggesting you can get 320mb of data from an entire run/picotiterplate.

However, the table in Roche technical bulletin TCB-09008 seems to suggest you only get 100mb per run when using the 8kb-library.

Can anyone guide as to the reason for the discrepancy?

Cheers

Nick.

[kmcarr]

Nick,

Yes, that bulletin does seem a bit confusing. But my read on that whole section is that they want you to prepare several different PE libraries from the same starting material and collect a limited amount of sequence from each of those libraries to avoid oversampling a single library. I interpret the 100Mbp number as the amount of data you should collect from any single 8kbp span library, not how much a run would produce. They do say in that section that for small genomes (<5Mbp) a single 8kbp library is sufficient to yield enough data.

[nickloman]

Nick,

Yes, that bulletin does seem a bit confusing. But my read on that whole section is that they want you to prepare several different PE libraries from the same starting material and collect a limited amount of sequence from each of those libraries to avoid oversampling a single library. I interpret the 100Mbp number as the amount of data you should collect from any single 8kbp span library, not how much a run would produce. They do say in that section that for small genomes (<5Mbp) a single 8kbp library is sufficient to yield enough data.

Thanks. Makes sense. So should I assume that a plate of 8kb paired-end produces the same number of reads as a standard WGS run? I realise throughput will be down a bit because of having to sequence the linker each time.

[RCJK]

Thanks. Makes sense. So should I assume that a plate of 8kb paired-end produces the same number of reads as a standard WGS run? I realise throughput will be down a bit because of having to sequence the linker each time.

Hi,

I think your assumption is correct. You'd have the same number of reads, just less sequence of interest due to the linkers.

Just curious if you've already done the 8Kb libraries for the small genome and if so, how'd it go?

[nickloman]

We're preparing a library at the moment and hope to run it this month, so I'll report back when we get the results ...