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Old 04-05-2017, 06:20 PM   #1
dladuswj
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Location: South Korea

Join Date: Apr 2017
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Post Problems of assembly polishing with RS_Resequencing.1 protocol

Hi all,

Iím experiencing a difficulty in getting a complete bacterial genome from PacBio sequencing data using the SMRT Analysis protocols.
My usual workflow is as follows:
De novo assembly by HGAP2 ->Circularization by circlator or minimus -> Polishing by RS_Resequencing.1 protocol until no variants are detected.
This workflow has worked nicely. Usually, repeating RS_Resequencing several times resulted in 100.0% of Consensus Concordance and no variants.

But, for a new bacterial genome we sequenced recently, the result of RS_Resequencing is different from usual. During repeating the RS_Resequencing 8 times, variants have been detected continuously at 10-20 positions and Consensus Concordance never reached 100.0%. At some positions, 1-bp insertion occurred repeatedly per each resequencing.


In my opinion, it seems that more repetition of RS_Resequencing would not be helpful.
What should I do to solve this problem?
Does anyone have a similar experience?

Thanks in advance!
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Old 04-06-2017, 11:07 AM   #2
gconcepcion
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Hi dladuswj,

It seems likely that the bacterial genome that you are sequencing contains more/longer homopolymer/repetitive regions than the typical genomes you are sequencing, which could result in further iterations of the Resequencing algorithm "failing to converge" on a 100% consensus.

You should investigate the alignments at the particular areas where the variants are being called to determine what is causing the variant calling to differ from round to round.
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Old 04-06-2017, 11:10 AM   #3
rhall
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It is possible that the data is from a population that is not perfectly clonal. This happens frequently in slow growing organisms, or things that mutate rapidly.
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