Combine TruSeq DNA PCR-Free library and 2x300bp PE sequencing on MiSeq

Johnny99 · 07-09-2015, 11:47 AM

Hi everyone,

We are about to do whole genome sequencing of an 12Mb organism. There are still a lot of "loose ends" in the reference genome, so we aim for long read lengths in order to contribute to the assembly.

Does anyone have experience in 2 x 300bp PE sequncing on the MiSeq, using the TruSeq DNA PCR-Free Library Prep kit (550bp fragments)? Our concern is that the shortest fragments hybridize more easily to the flowchip causing a lot of redundant reads. There will of course be an overlap of 50bp for the 550bp fragments, but will the problem of big overlaps be extensive?

Has anyone used alternative protocols with a more strict size selection or adjusted settings for the fragmentation to get less short reads?

Any help is greatly appreciated!

nucacidhunter · 07-09-2015, 04:26 PM

If you use standard bead-based size selection approximately half of reads will have length below 550 bp (see PCR-free user guide plots). Two options:

1- Use more input DNA for PCR-free and size-select with Pippin and follow the rest of protocol
2- PacBio, with 12Mb genome you can get 100x coverage with 2-3 SMRT cells which would give better results than any Illumina system for less cost

kerplunk412 · 07-16-2015, 06:23 PM

Quote:

Originally Posted by nucacidhunter

2- PacBio, with 12Mb genome you can get 100x coverage with 2-3 SMRT cells which would give better results than any Illumina system for less cost

I completely agree with this. If you are looking to "finish" a genome I believe PacBio is generally accepted to be the gold standard.

Brian Bushnell · 07-16-2015, 09:23 PM

Quote:

Originally Posted by kerplunk412

I completely agree with this. If you are looking to "finish" a genome I believe PacBio is generally accepted to be the gold standard.

That's absolutely true. Pure PacBio is currently the cheapest and most effective way to produce a high-quality single bacterial genome. That said, I've never heard of a prokaryote with a 12Mbp genome... so I'd really need to know what this organism is before giving a firm recommendation.

Aside from PacBio, you can still get an extremely good assembly for most bacteria using Illumina 2x300 reads. Depending on the assembler, it may be useful to merge reads before assembling. You will not get a 1-contig assembly from an Illumina fragment-only library on a bacteria, regardless of the insert size, but the result will typically still be very good for most purposes.

Johnny99 · 08-03-2015, 01:57 AM

Thanks a lot for your considerations!

The organism is an eukaryotic parasite. Our primary goal for the experiment is variant calling, so we aim for good coverage. However, it would be a bonus if we could contribute to improve the reference genome at the same time. That is why we consider long reads. That might be too much to ask for...

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
TruSeq DNA PCR-Free Sample Preparation Kit (Illumina) expired 3 months - can I use?	snarapor	Sample Prep / Library Generation	3	08-22-2015 06:59 AM
Illumina TruSeq PCR-Free - Low Final Concentration WGS Rat DNA Libraries	agileta	Illumina/Solexa	1	11-18-2014 08:11 PM
TruSeq DNA PCR-free vs NANO DNA kit	mbk0asis	Illumina/Solexa	8	05-17-2014 09:15 AM
Truseq DNA PCR-free kit	Laruo	Sample Prep / Library Generation	1	03-05-2014 03:42 AM
The NEXTflex™ PCR-Free DNA Sequencing Kit Reduces Bias in NGS Library Prep	Bioo Scientific	Vendor Forum	10	12-20-2012 08:10 AM

07-09-2015, 11:47 AM	#1
Johnny99 Junior Member Location: Norway Join Date: Jul 2015 Posts: 2	Combine TruSeq DNA PCR-Free library and 2x300bp PE sequencing on MiSeq Hi everyone, We are about to do whole genome sequencing of an 12Mb organism. There are still a lot of "loose ends" in the reference genome, so we aim for long read lengths in order to contribute to the assembly. Does anyone have experience in 2 x 300bp PE sequncing on the MiSeq, using the TruSeq DNA PCR-Free Library Prep kit (550bp fragments)? Our concern is that the shortest fragments hybridize more easily to the flowchip causing a lot of redundant reads. There will of course be an overlap of 50bp for the 550bp fragments, but will the problem of big overlaps be extensive? Has anyone used alternative protocols with a more strict size selection or adjusted settings for the fragmentation to get less short reads? Any help is greatly appreciated!

07-09-2015, 04:26 PM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	If you use standard bead-based size selection approximately half of reads will have length below 550 bp (see PCR-free user guide plots). Two options: 1- Use more input DNA for PCR-free and size-select with Pippin and follow the rest of protocol 2- PacBio, with 12Mb genome you can get 100x coverage with 2-3 SMRT cells which would give better results than any Illumina system for less cost

08-03-2015, 01:57 AM	#5
Johnny99 Junior Member Location: Norway Join Date: Jul 2015 Posts: 2	Thanks a lot for your considerations! The organism is an eukaryotic parasite. Our primary goal for the experiment is variant calling, so we aim for good coverage. However, it would be a bonus if we could contribute to improve the reference genome at the same time. That is why we consider long reads. That might be too much to ask for...