Ion Torrent Reference Assembly

[jdilts]

in 7 days

In the future me know if you have any programming issues. I'd be glad to help you out.

[hengnck]

The script isn't too complicated. It would be something similar to this. I hope this can be of some assistance.

#/usr/bin/perl

use strict;
use warnings;

my $infile ="readFILE";
my $outfile = "quality_readsFILE";

#opens file with reads
open (IN,<,$infile) || die $!;
my @reads = <IN>; #stores each line in the file into an arrary
close (IN); #don't need the file anymore, close it


open (OUT,>,$outfile) || die $!; #open the out going file

my $j = 0; #array index
my $read_name;

#iterate through array
foreach my $i (@reads){
	if ($j%2 = 1) && (length($i)>=75){
	print OUT "$read_name\n$i\n";}
	}
	else{ $read_name = $i;} #stores read name
}
close (OUT);

Thanks, jdilts. Will try things out - as soon as I kill the MIRA assembly (7 days and counting).

[jdilts]

Not too complicated

The script isn't too complicated. It would be something similar to this. I hope this can be of some assistance.

#/usr/bin/perl

use strict;
use warnings;

my $infile ="readFILE";
my $outfile = "quality_readsFILE";

#opens file with reads
open (IN,<,$infile) || die $!;
my @reads = <IN>; #stores each line in the file into an arrary
close (IN); #don't need the file anymore, close it


open (OUT,>,$outfile) || die $!; #open the out going file

my $j = 0; #array index
my $read_name;

#iterate through array
foreach my $i (@reads){
	if ($j%2 = 1) && (length($i)>=75){
	print OUT "$read_name\n$i\n";}
	}
	else{ $read_name = $i;} #stores read name
}
close (OUT);

[hengnck]

Re: Downsampling

What size do you want the reads? You could cut out all the smaller reads. (Maybe 50bp or less?) To do this you would need to write up a script of some sort. Either perl or python to get the length of each read and then output the reads that "qualify" into an outfile.

@jdilts - OK, I understand - I need to go see a perl or python programmer. The max read length was 398 but average was 177. My collaborator who did the sequencing did not specify the type of kit used but they're all shotgun reads. I will have to play around with min and max reads.

[jdilts]

What size do you want the reads? You could cut out all the smaller reads. (Maybe 50bp or less?) To do this you would need to write up a script of some sort. Either perl or python to get the length of each read and then output the reads that "qualify" into an outfile.

[hengnck]

Downsampling ion data

Hi All,

Thanks for your comments - you can all probably see that I'm more comfortable in the Sanger era. Unfortunately in my Faculty, I'm the "bioinformatics team".

My default SOP is to use all the data - the more the merrier - but I can see now that I've got way too much data than required. How do I downsample 480 Mbp of essentially random reads down to 100-150 Mbp?

All advice is greatly appreciated.

[kmcarr]

I concur with IT's comments about excessive coverage. You really need to scale back your input to ~30X. Also why Newbler 2.3? That is a very old version. Get version 2.6, they have made several improvements in the assembler.

[IonTorrent]

Hi - I'm working with a small bacterial genome ~2.0 Mbp but de novo (new species). Got data from an Ion 318 chip, about 480 Mbp. Ran it through Newbler 2.3 - 3,000+ contigs. Set up default MIRA assembly *six* days ago and it's still going. :-( I wouldn't use DNA* - ridiculously expensive for what it does. Roche RefMapper is OK for some of our other known bacterial genomes.

Hi hengnck,

Are you using all 480 Mbp of data in the assembly or are you downsampling? I ask because many software packages (like those you mention) will grossly underperform with excessive coverage, and are reported to work best in the 30X to 50X range (and if this is DNA from pure culture you're at ~240X). Are you a Torrent Suite user and if so are you using the MIRA plugin? The newest (v2.2) version allows you to specify the amount of coverage to use (best results are typically see at ~50X):

http://lifetech-it.hosted.jivesoftware.com/docs/DOC-2572

Some have commented that they use Newbler at around 30X coverage for de novo assembly.

[RonanC]

I just noticed that CLC bio are offering a free 6 month trial of their CLC genomics workbench to users with a benchtop NGS (i.e. 454 GS Jr, MiSeq or IonTorrent PGM). Anybody have any experience with the CLC software?

[hengnck]

Hi - I'm working with a small bacterial genome ~2.0 Mbp but de novo (new species). Got data from an Ion 318 chip, about 480 Mbp. Ran it through Newbler 2.3 - 3,000+ contigs. Set up default MIRA assembly *six* days ago and it's still going. :-( I wouldn't use DNA* - ridiculously expensive for what it does. Roche RefMapper is OK for some of our other known bacterial genomes.

[jdilts]

Is anyone using Newbler, DNASTAR, MIRA?

[jdilts]

What is the best reference assembler to use with ion torrent data? I can only seem to find information on Ion Torrent de novo assemblies, which is not what I'm looking for. Thanks in advance!