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Old 03-13-2018, 10:29 PM   #1
wanadou
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Default help for NGS and assembly

Hello

I think to do the whole genome sequencing of a halophyte plant (350 Mbp) using the Hiseq Pe150 which generate data with 30X coverage
1- this strategy could it be effective to make an de novo assembly in order to have a draft genome?
or
the sequencing using PacBio (1 cell SMRT) to generate sequences with more bigger size is it effective to make de novo assembly of the draft genome?
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Old 03-14-2018, 02:38 AM   #2
nucacidhunter
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The genome size is relatively small so you will have better assembly if you sequence with PacBio but one SMRT cell will not be enough. You should consider 50x coverage (17.5 Gb) which will require 3-4 Sequel SMRT cells. You can also sequence 2 SMRT cells and then do a preliminary assembly and decide on further sequencing.

Illumina sequencing will result in lots of short contigs especially if the plant genome is composed of repeat regions.

I do not think any of these platforms will enable complete assembly but the results will be better if DNA is from a homozygous plant.
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Old 03-14-2018, 11:16 PM   #3
wanadou
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Hello nucacidhunter,
Thanks for your reply
The cost of sequencing using PacBio is relatively high. the sequencing of 1 cell SMRT can give 5Gb. I think to do an preliminary assembly for a draft genome. In second time i will do sequencing using Illumina platform which provide 50X coverage. this second sequencing can be used to do hybrid assembly in ordre to have complete genome.
you think this strategy is feasible? or 10-15X coverage given by PacBio is insuffusant to do draft genome assembly
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Old 03-15-2018, 01:26 AM   #4
nucacidhunter
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It depends on the genome. You might look at repeat content of a related species if it is available. I have seen Illumina based assembled contigs which were couple of hundred bases larger than the sequencing length.

I do not think either of these platforms alone or in combination will give complete assembly but I would recommend to do 2 SMRT cells and assemble then depending on results decide for more sequencing on one. For instance, if you are interested in particular genes or region covered by PacBio contigs then Illumina will be good to increase accuracy. Depending on your application an incomplete assembly might be enough.

PacBio is sensitive to carry over of contaminants from DNA extraction so you should try an extraction method that result in long fragments and is free from carbohydrates and phenolic compounds. Link for PacBio calculator:
https://www.pacb.com/cn/calculator-w...me-sequencing/
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Old 04-16-2018, 06:10 AM   #5
colindaven
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Its' very tricky to analyse 10X pacbio data and do a de novo assembly. I have had very bad experiences in the past with this setup, getting poor N50s and putting lots of effort in. 30x+ is far, far better since you need a lot of the longest reads to span gaps AND self correct reads for a decent assembly .

There were a few tools for low coverage pacbio hybrid assembly, DBG2OLC for example
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Old 04-16-2018, 08:13 AM   #6
Markiyan
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Lightbulb Use Illumina 2x250 or 2x300, not 2x150 in de novo!

Please use Illumina 2x250 or 2x300 in any de novo appication! (in addition to the pacbio).

It means 1-2 runs (2x250 or 2x300) using MiSeq or 1/2 of the HiSeq 2500 RR 2x250.

Usually it gives you significantly better assemblies than 2x150 or 2x100 bp runs.

PS: Make sure a PCR-free library prep is used.
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Old 04-17-2018, 04:37 PM   #7
gringer
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If you want cheap, [MinION](https://store.nanoporetech.com/basic.html) is always available (starter discount is $1000 for two flow cells and a rapid kit). The consensus assemblies are less accurate than PacBio, but if you're doing Illumina as well for local assembly correction (which I also recommend) that probably won't matter as much.
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