De novo genome assembly (Hybrid) - bacterial genome

MuG · 03-28-2017, 07:26 AM

Hi,

I would like to do an de novo genome assembly of a bacterial genome. I already have PacBio data and will now do Illumina sequencing, aditionally. I plan to do HiSeq, as I want to do a SNP analysis too. Which tool can a use to perform a hybrid assembly with PacBio and Illumina HiSeq data? PacBio gives me 5 contigs. Do you think that I can reduce the contigs with my HiSeq data?

Thank you in advance!!

Markiyan · 03-28-2017, 08:26 AM

A couple of points with respect of experimental design:

1. Make sure the Illumina libraries are PCR-free prep.
2. Unless you want to do 96 genomes on one hiseq lane, use MiSeq, since hiseq would give too much data (you would have to subsample), also to ensure that you do 2x250 or 2x300 read length.

So use Miseq (~10-16 samples per 2x250 or 2x300 run) or Hiseq2500 in 2x250 rapid run mode.

This would help the downstream analysis (pacbio error correction/assembly) quite a lot. (compared to 2x100 or 2x150 bps read length), especially in the repeat regions.

Tools:
Give spades assembler a try...

luc · 03-28-2017, 04:13 PM

Illumina data are very unlikely stitch your Pacbio contigs together. I would suggest to try different assemblers and parameters on your Pacbio data.

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03-28-2017, 07:26 AM	#1
MuG Junior Member Location: Austria Join Date: Mar 2017 Posts: 1	De novo genome assembly (Hybrid) - bacterial genome Hi, I would like to do an de novo genome assembly of a bacterial genome. I already have PacBio data and will now do Illumina sequencing, aditionally. I plan to do HiSeq, as I want to do a SNP analysis too. Which tool can a use to perform a hybrid assembly with PacBio and Illumina HiSeq data? PacBio gives me 5 contigs. Do you think that I can reduce the contigs with my HiSeq data? Thank you in advance!!

03-28-2017, 08:26 AM	#2
Markiyan Member Location: Cambridge Join Date: Sep 2010 Posts: 90	You probably want to use miSeq in 2x250 for data aquisition. A couple of points with respect of experimental design: 1. Make sure the Illumina libraries are PCR-free prep. 2. Unless you want to do 96 genomes on one hiseq lane, use MiSeq, since hiseq would give too much data (you would have to subsample), also to ensure that you do 2x250 or 2x300 read length. So use Miseq (~10-16 samples per 2x250 or 2x300 run) or Hiseq2500 in 2x250 rapid run mode. This would help the downstream analysis (pacbio error correction/assembly) quite a lot. (compared to 2x100 or 2x150 bps read length), especially in the repeat regions. Tools: Give spades assembler a try...

03-28-2017, 04:13 PM	#3
luc Senior Member Location: US Join Date: Dec 2010 Posts: 283	Illumina data are very unlikely stitch your Pacbio contigs together. I would suggest to try different assemblers and parameters on your Pacbio data.