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

Join Date: Apr 2017
Posts: 1
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!
dladuswj is offline   Reply With Quote
Old 04-06-2017, 11:07 AM   #2
Location: Menlo Park

Join Date: Dec 2010
Posts: 68

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.
gconcepcion is offline   Reply With Quote
Old 04-06-2017, 11:10 AM   #3
Senior Member
Location: San Francisco

Join Date: Aug 2012
Posts: 318

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.
rhall is offline   Reply With Quote

pacbio, rs_resequencing.1

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