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Old 01-10-2013, 03:30 AM   #1
RGLADSTONE
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Default Miseq assemblies

Bacteria sequencing. Nextera XT library prep. Triplicate technical replicates from a single MiSeq run 2 x 250 PE, de novo assembly on MiSeq gives ~450 contigs for all three. Optimized velvet assembly from fastq's gives 200 contigs for two samples and 800 contigs for one. Why would there be so much of a difference between 3 technical replicates for one method of assembly and not for the other?
To make things more complicated the triplicate samples are from a single strain but three individual DNA extractions.

Last edited by RGLADSTONE; 01-11-2013 at 02:28 AM.
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Old 01-11-2013, 03:25 AM   #2
mwatson
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Default

Quote:
Originally Posted by RGLADSTONE View Post
Bacteria sequencing. Nextera XT library prep. Triplicate technical replicates from a single MiSeq run 2 x 250 PE, de novo assembly on MiSeq gives ~450 contigs for all three. Optimized velvet assembly from fastq's gives 200 contigs for two samples and 800 contigs for one. Why would there be so much of a difference between 3 technical replicates for one method of assembly and not for the other?
To make things more complicated the triplicate samples are from a single strain but three individual DNA extractions.
1) is there a reference? If so, map and then estimate the insert size
2) Try FLASH to see if the reads overlap
3) Trim to 100bp PE and assemble again -> does this help?
4) Randomly choose 10%, 25%, 50% of the data, assemble again, does this help?
5) have you checked for adapters?
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assembly, illumina miseq, velvet paired-end

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