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naleph 02-08-2017 09:10 AM

Uneven coverage across scaffold

I have run the SMRT-Portal RS_HGAP_Assembly.2 protocol.
I am getting a single whole chromosome assembled (~4.4Mb) but there is uneven coverage across the scaffold.

See Figure:

Additionaly the start 15Kb and the 15Kb where the coverage drops is a repeat.

I am unsure how to interperate these results and I am wondering if there has been a misassembly.

Could anyone shed some light on this?

Brian Bushnell 02-08-2017 09:15 AM

You appear to have a 750kb collapsed repeat at the beginning, which is why the coverage is doubled.

naleph 02-09-2017 02:50 AM

I had thought of this scenario but duplication of almost a quarter of the genome is highly unlikely and illumina sequencing produces even coverage across the genome. Is it possible that a large segment of the genome was unevenly sequenced with PacBio?

Brian Bushnell 02-09-2017 09:28 AM

Could be a plasmid with two copies, or a plasmid that replicates part of the main chromosome. PacBio does not normally produce uneven coverage; it's very flat. Can you post a picture of the Illumina coverage?

naleph 02-10-2017 02:42 AM

Sure! here it is:

Also the organism doesn't have a plasmid.
Thanks for your help.

SylvainL 02-10-2017 03:48 AM

Is it supposed to be a linear or circular genome?

naleph 02-10-2017 04:18 AM

The genome is Circular

HESmith 02-10-2017 06:22 AM

1) What is the organism that you're studying? And are there any related genomes that might be useful for comparison?

2) Is there any possibility that the sample might be heterogeneous/contaminated, and that the 750kbp segment is from a different species or symbiont? It might be informative to compare the GC content of that segment to the remainder of the chromosome. Kmer plots and BLAST searches might also prove useful.

3) And any chance that the Illumina dataset was normalized?

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