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-   -   Ion torrent: bacterial WGS coverage (http://seqanswers.com/forums/showthread.php?t=31365)

roshanbernard 06-25-2013 07:20 AM

Ion torrent: bacterial WGS coverage
 
Hi all,

I am new to ion torrent.

We are interested in getting the bacterial whole genome sequencing( ~2MB )...

However, i am not sure what is the coverage/depth we need to use to get the good read quality and assembly.

Can anyone help me out with this

regards

rosh

krobison 06-25-2013 08:37 AM

De novo or resequencing?

Probably shoot for >50X coverage minimum. Higher initial coverage will allow more aggressive trimming/filtering of reads.

I have not assembled Ion data for a while, but historically the homopolymer calling issue leads to assemblies rich in indels. Perhaps someday someone will write the equivalent of Quiver for Ion Torrent data, but that hasn't happened yet.

Quality of the assembly will also be significantly affected by GC content (expect issues in regions with extremes in GC content) and the nature of the repeats in the genome. Set your expectations appropriately; short read, short insert technologies such as Ion cannot routinely close bacterial genomes -- you need either very long reads (PacBio) or mate pairs to have good odds of doing that.

roshanbernard 06-25-2013 04:47 PM

ah okie... previously we have used the illumina reads. this is the first time we are going with ion torrent. anyways lets hope for the best.. i am planning to go with 35 to 40X... thanks a lot..

ajthomas 06-26-2013 08:11 AM

You might consider doing one run on a MiSeq or something and mixing that data with your Ion Torrent data to correct the indels. You might also consider using 454 instead of Ion Torrent since the longer reads will aid in assembly. 1/8 of a plate would give you the coverage you're looking for.

krobison 06-26-2013 11:19 AM

If you are considering other platforms, for about half the price (based on looking at one core lab's prices) of the 454 data you could run only Pacific Biosciences and almost certainly get a superior assembly.

How many strains are you sequencing & what are you estimating for the full cost per strain (library prep, sequencing) on Ion Torrent? On PacBio, your genome should be <$700 each.

roshanbernard 06-26-2013 08:07 PM

My boss is interested in ion torrent.. because we have got that machine in our institute.. however yesterday we spoke to applied biosciances, they said they are gonna use 318chips with 100X coverage... dont know if i can get the proper aqssembly. i use geneious for the assembly....

jonathanjacobs 07-01-2013 04:19 AM

For what it's worth - higher coverage will NOT not lead to better de novo assemblies. Once you hit about 40-50x , higher coverage may actually produce worse assemblies. Using an Ion318 for a 2MB genome is IMHO a waste of a chip and funding. You would be much better off running a 316 or even a 314 (314's in our lab routinely return 50-60mb of data, more than enough for a 2mb genome) and then using the money you save to outsource a PacBio or Illumina MiSeq run and then do a hybrid assembly.

That's my 2

roshanbernard 07-01-2013 05:24 PM

Quote:

Originally Posted by jonathanjacobs (Post 109157)
For what it's worth - higher coverage will NOT not lead to better de novo assemblies. Once you hit about 40-50x , higher coverage may actually produce worse assemblies. Using an Ion318 for a 2MB genome is IMHO a waste of a chip and funding. You would be much better off running a 316 or even a 314 (314's in our lab routinely return 50-60mb of data, more than enough for a 2mb genome) and then using the money you save to outsource a PacBio or Illumina MiSeq run and then do a hybrid assembly.

That's my 2

thanks a lot jonathan. Our preference was to save money. we are not interested in gap filling and stuff. we were looking for some specific genes. we have 84 bacterial samples for sequencing. Ur info is helpful. thanks a lot.


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