Shotgun metagenomics study for environmental samples using PacBio

weigrc · 09-20-2017, 12:20 AM

Dear all,

Anyone has such experience? How to conquer the issue of low input DNA?

Thanks,
Wei

Markiyan · 09-20-2017, 04:08 AM

Two points:

1. To get more DNA try making the sample prep more efficient/use more starting material/

2. To allow efficient error-correction of the pacbio reads also prep PCR-free (optional: + Nextera matepair) libraries from the same lot of DNA as used for creating the PAC-Bio libraries, and sequence them at 30X coverage compared to the volume of the raw pacbio data you are getting.

First do self-errorcorection of the pacbio subreads from the same template,
than error correct the filtered pacbio subreads using the Illumina data before the assembly.

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09-20-2017, 12:20 AM	#1
weigrc Member Location: Hong Kong Join Date: Oct 2011 Posts: 46	Shotgun metagenomics study for environmental samples using PacBio Dear all, Anyone has such experience? How to conquer the issue of low input DNA? Thanks, Wei

09-20-2017, 04:08 AM	#2
Markiyan Senior Member Location: Cambridge Join Date: Sep 2010 Posts: 116	Make sure to have Illumina data from the same DNA prep for efficient error-correction Two points: 1. To get more DNA try making the sample prep more efficient/use more starting material/ 2. To allow efficient error-correction of the pacbio reads also prep PCR-free (optional: + Nextera matepair) libraries from the same lot of DNA as used for creating the PAC-Bio libraries, and sequence them at 30X coverage compared to the volume of the raw pacbio data you are getting. First do self-errorcorection of the pacbio subreads from the same template, than error correct the filtered pacbio subreads using the Illumina data before the assembly.