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Re: [wgs-assembler-users] wgs-assembler-users Digest, Vol 3, Issue 1
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From: Waldbieser, G. <Geoff.Waldbieser@ARS.USDA.GOV> - 2012-06-13 20:46:29
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When producing a mer database from a set of paired end Illumina reads, you recommend a lower limit of 15X genome coverage. Is there an upper limit to the amount of genome coverage so that one does not oversample sequencing error?
Geoff Waldbieser
-----Original Message-----
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Sent: Tuesday, June 12, 2012 12:39 AM
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Subject: wgs-assembler-users Digest, Vol 3, Issue 1
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Today's Topics:
1. FastqToCA for paired-end reads (Mundy, Michael)
2. Re: FastqToCA for paired-end reads (Ole Kristian T?rresen)
3. Re: small gaps of fixed length (Sajeet Haridas)
4. Ion torrent data (Powers, Jason)
5. ContigContainment failed (Ole Kristian T?rresen)
6. Re: ContigContainment failed (Walenz, Brian)
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Message: 1
Date: Mon, 14 May 2012 13:32:19 -0500
From: "Mundy, Michael" <Mun...@ma...>
Subject: [wgs-assembler-users] FastqToCA for paired-end reads
To: <wgs...@li...>
Message-ID: <CBD6B9E3.978%Mun...@ma...>
Content-Type: text/plain; charset="iso-8859-1"
I?m using WGS 7.0 and I have two synchronized fastq files with paired-end
reads. Based on the documentation at
http://sourceforge.net/apps/mediawiki/wgs-assembler/index.php?title=FastqToC
A, I tried this command:
wgs-7.0/Linux-amd64/bin/fastqToCA -libraryname SRR067601.000 -mates
SRR067601.000_1_pair.fq,SRR067601.000_2_pair.fq
But it returns this error:
ERROR: Mated reads (-mates) must have am insert size (-insertsize).
The documentation page says that the ?insertsize option is optional so I
thought that was the flag to distinguish between paired-end reads and
mate-pair reads. How do I generate a FRG file for paired-end reads?
Mike Mundy
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Message: 2
Date: Mon, 14 May 2012 20:46:31 +0200
From: Ole Kristian T?rresen <o.k...@bi...>
Subject: Re: [wgs-assembler-users] FastqToCA for paired-end reads
To: "Mundy, Michael" <Mun...@ma...>
Cc: wgs...@li...
Message-ID:
<CAH...@ma...>
Content-Type: text/plain; charset=windows-1252
On 14 May 2012 20:32, Mundy, Michael <Mun...@ma...> wrote:
> I?m using WGS 7.0 and I have two synchronized fastq files with paired-end
> reads. ?Based on the documentation at
> http://sourceforge.net/apps/mediawiki/wgs-assembler/index.php?title=FastqToCA,
> I tried this command:
>
> wgs-7.0/Linux-amd64/bin/fastqToCA -libraryname SRR067601.000 -mates
> SRR067601.000_1_pair.fq,SRR067601.000_2_pair.fq
>
> But it returns this error:
>
> ERROR: ?Mated reads (-mates) must have am insert size (-insertsize).
>
> The documentation page says that the ?insertsize option is optional so I
> thought that was the flag to distinguish between paired-end reads and
> mate-pair reads. ?How do I generate a FRG file for paired-end reads?
I guess the documentation is not up to date, so it's not optional to
supply the -insertsize option. Just add -insertsize 300 30, if your
reads are from a 300 bp DNA fragment and are paired end, or do
something like -insertsize 5000 500 -outtie if they are mate pairs
from a 5k library.
Ole
>
> Mike Mundy
>
>
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------------------------------
Message: 3
Date: Fri, 18 May 2012 11:26:28 -0700
From: "Sajeet Haridas" <sa...@gm...>
Subject: Re: [wgs-assembler-users] small gaps of fixed length
To: <wgs...@li...>
Message-ID: <000601cd3523$bed2dcb0$3c789610$@com>
Content-Type: text/plain; charset="us-ascii"
Hello Brian,
Since all the CTP mea is more than -20, can I assume that no overlaps were
skipped where contigs overlapped more than 20 bases (ie: all overlaps > 20
were successfully merged into scaffolds with no N's).
How does the scaffolder behave when the overlap between contgs is 5bp or
less?
For a small fungal genome with relatively little repeats and no allelic
variations (is haploid), are there any parameters the can reduce the number
of false gaps. Will increasing cgwErrorRate help?
Thank you,
Sajeet
-----Original Message-----
From: Walenz, Brian [mailto:bw...@jc...]
Sent: May-16-12 7:35 PM
To: Sajeet Haridas
Subject: RE: small gaps of fixed length
IIRC, that's the marker for "contigs should overlap, but no overlap found".
Possibilities here: the overlap is shorter than we can detect, or there is
crud on the end of one contig, or the error rate is too high.
Are you on the mailing list? This would have been a nice discussion there.
________________________________________
From: Sajeet Haridas
Sent: Wednesday, May 16, 2012 8:22 PM
To: Walenz, Brian
Subject: RE: small gaps of fixed length
Thank you Brian.
I also notice that the minimum CTP mea is -20. Is this value also capped?
Sajeet
From: Walenz, Brian
Sent: May-16-12 1:12 PM
To: Sajeet Haridas
Subject: Re: small gaps of fixed length
Yes - that's the lower limit on a gap between contigs. Either mate pairs
indicate the contigs should overlap but no overlap could be found, or there
really is a small positive gap. Only the asm file will distinguish the two.
Look under 'mea' here:
http://sourceforge.net/apps/mediawiki/wgs-assembler/index.php?title=ASM_File
s#SCF_CTP
b
On 5/16/12 3:29 AM, "Sajeet Haridas" wrote:
Hello Brian,
My fungal genome assemblies (30-35MBp) always seem to have ~2500 small gaps
always represented by 20 N's - using bog, bogart and various other
parameters.
Is the assembler trying to tell me something?
Thank you,
Sajeet
------------------------------
Message: 4
Date: Sun, 20 May 2012 13:09:47 +0000
From: "Powers, Jason" <jp...@ex...>
Subject: [wgs-assembler-users] Ion torrent data
To: "wgs...@li..."
<wgs...@li...>
Message-ID:
<1EF140AF181DEF48AAAB3C8A832AE432010AC1AD@EA-EXCHANGE3.ExpressionAnalysis.local>
Content-Type: text/plain; charset="us-ascii"
Any thoughts about the best settings in fastqToCA for Ion Torrent data? My guess is that it is closest to illumina, but thought I would see what you guys thought.
Thanks
Jason
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Message: 5
Date: Mon, 11 Jun 2012 19:51:27 +0200
From: Ole Kristian T?rresen <o.k...@bi...>
Subject: [wgs-assembler-users] ContigContainment failed
To: wgs...@li...
Message-ID:
<CAH...@ma...>
Content-Type: text/plain; charset=ISO-8859-1
Hi,
I got an assertion fail while scaffolding today. Error message:
CreateNewGraphNode()-- Contig 16085537
* Create a contig 16085537 in scaffold 131913
>>> Fixing up suspicious overlap (15370134,16085537,I) (ahg:-340 bhg:-143) to (15370134,16085537,O) (ahg:143 bhg:340) len: 137
* FOEXS: SUSPICIOUS Overlap found! Looked for
(16085537,15370134,I)[20,1044] found (15370134,16085537,O) 137
WARNING: InsertChunkOverlap()-- Chunk overlap already exists.
NEW 15370134,16085537,O - min/max 131/142 0/0 erate 0.100000 flags
10000 overlap 137 hang 0,0 qual 0.000000 offset 0,0
OLD 15370134,16085537,O - min/max 20/1044 20/1044 erate 0.100000 flags
10001 overlap 137 hang 143,340 qual 0.000000 offset 0,0
WARNING: CreateChunkOverlapFromEdge()-- Chunk overlap already exists.
Keeping old overlap.
NEW 15370134,16085537,O - min/max 131/142 0/0 erate 0.100000 flags
10000 overlap 137 hang 0,0 qual 0.000000 offset 0,0
OLD 15370134,16085537,O - min/max 20/1044 20/1044 erate 0.100000 flags
10001 overlap 137 hang 143,340 qual 0.000000 offset 0,0
* Switched right-left, orientation went from I to O
* CreateAContigInScaffold() failed.
ContigContainment failed.
cgw: LeastSquaresGaps_CGW.C:1410: RecomputeOffsetsStatus
RecomputeOffsetsInScaffold(ScaffoldGraphT*, CDS_CID_t, int, int, int):
Assertion `0' failed.
This is about 30x coverage with Illumina reads; 10x combined reads
(180 bp insert, 100 nt reads and combined with FLASH), 10x PE 100 nt
reads 300 insert and 5k mate pair 100 nt reads, all error corrected
with Quake. It's probably not an optimal combination, but I'm testing
a bit and interested in the result.
Using bogart and dnc, the 5k library compared against the other two.
Other options are default.
Is that assertion something that can be fixed?
Thank you.
Ole
------------------------------
Message: 6
Date: Tue, 12 Jun 2012 01:38:29 -0400
From: "Walenz, Brian" <bw...@jc...>
Subject: Re: [wgs-assembler-users] ContigContainment failed
To: Ole Kristian T?rresen <o.k...@bi...>,
"wgs...@li..."
<wgs...@li...>
Message-ID: <CBFC4E15.4865%bw...@jc...>
Content-Type: text/plain; charset="iso-8859-1"
Hi, Ole-
After merging two scaffolds, we do a least squares estimate of the gap sizes
in the new scaffold. If those gap sizes imply two contigs should be merged
(via a negative gap) we try to merge them. This merge failed for reasons I
haven't looked into. Probably bad sequence alignment.
I think you can safely disable this assert. Gap size estimation might run
through a few more iterations (with the same result) and eventually give up.
The scaffold will have (slightly?) bogus gap sizes. There are,
unfortunately, plenty of other scaffolds that fail to get gap size
estimates, so this isn't a disaster.
b
On 6/11/12 1:51 PM, "Ole Kristian T?rresen" <o.k...@bi...> wrote:
> Hi,
> I got an assertion fail while scaffolding today. Error message:
> CreateNewGraphNode()-- Contig 16085537
> * Create a contig 16085537 in scaffold 131913
>>>> Fixing up suspicious overlap (15370134,16085537,I) (ahg:-340 bhg:-143) to
>>>> (15370134,16085537,O) (ahg:143 bhg:340) len: 137
> * FOEXS: SUSPICIOUS Overlap found! Looked for
> (16085537,15370134,I)[20,1044] found (15370134,16085537,O) 137
> WARNING: InsertChunkOverlap()-- Chunk overlap already exists.
> NEW 15370134,16085537,O - min/max 131/142 0/0 erate 0.100000 flags
> 10000 overlap 137 hang 0,0 qual 0.000000 offset 0,0
> OLD 15370134,16085537,O - min/max 20/1044 20/1044 erate 0.100000 flags
> 10001 overlap 137 hang 143,340 qual 0.000000 offset 0,0
> WARNING: CreateChunkOverlapFromEdge()-- Chunk overlap already exists.
> Keeping old overlap.
> NEW 15370134,16085537,O - min/max 131/142 0/0 erate 0.100000 flags
> 10000 overlap 137 hang 0,0 qual 0.000000 offset 0,0
> OLD 15370134,16085537,O - min/max 20/1044 20/1044 erate 0.100000 flags
> 10001 overlap 137 hang 143,340 qual 0.000000 offset 0,0
> * Switched right-left, orientation went from I to O
> * CreateAContigInScaffold() failed.
> ContigContainment failed.
> cgw: LeastSquaresGaps_CGW.C:1410: RecomputeOffsetsStatus
> RecomputeOffsetsInScaffold(ScaffoldGraphT*, CDS_CID_t, int, int, int):
> Assertion `0' failed.
>
>
> This is about 30x coverage with Illumina reads; 10x combined reads
> (180 bp insert, 100 nt reads and combined with FLASH), 10x PE 100 nt
> reads 300 insert and 5k mate pair 100 nt reads, all error corrected
> with Quake. It's probably not an optimal combination, but I'm testing
> a bit and interested in the result.
>
> Using bogart and dnc, the 5k library compared against the other two.
> Other options are default.
>
> Is that assertion something that can be fixed?
>
> Thank you.
>
> Ole
>
> ------------------------------------------------------------------------------
> Live Security Virtual Conference
> Exclusive live event will cover all the ways today's security and
> threat landscape has changed and how IT managers can respond. Discussions
> will include endpoint security, mobile security and the latest in malware
> threats. http://www.accelacomm.com/jaw/sfrnl04242012/114/50122263/
> _______________________________________________
> wgs-assembler-users mailing list
> wgs...@li...
> https://lists.sourceforge.net/lists/listinfo/wgs-assembler-users
------------------------------
------------------------------------------------------------------------------
Live Security Virtual Conference
Exclusive live event will cover all the ways today's security and
threat landscape has changed and how IT managers can respond. Discussions
will include endpoint security, mobile security and the latest in malware
threats. http://www.accelacomm.com/jaw/sfrnl04242012/114/50122263/
------------------------------
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