[pari_89]

Hi - has anybody tried referenced or de novo assembly from the .sam (or .fastq) files and ion Torrent datasets ? I downloaded the following data sets from the ion Community:

B7-143
B7-295
C19-543

And I do have .bam (.sam) and .fastq files

Velvet de novo assembly technically worked, but left me with many thousands of small contigs, so it is useless

(*) Velvet

All trials of referenced assembly with the Columbus extension to Velvet and Mosaik Assembler (using sorted sam files) and Mosaik Assembler (MosaikBuild) failed, apparently for serious inconsistencies on the data set or data format incompatibility:
[0.000000] Reading FastA file NC_010473.C19-543.genome.fasta;
[59.568244] 1 sequences found
[59.568247] Done
[59.568619] Reading SAM file C19-543.sorted.sam
[355.404283] 6906611 reads found.
[355.404285] Done
[355.404286] Reference mapping counters
[355.404287] Name Read mappings
[355.404288] gi|170079663|ref|NC_010473.1| 19688393
[356.782277] Reading read set file C19-543/Sequences;
[363.732809] 6906612 sequences found
[363.733517] Read 1 of length 32794, longer than limit 32767
[363.733519] You should modify recompile with the LONGSEQUENCES option (cf. manual)

(*) MosaikBuild

MosaikBuild -q B7-295.fastq.gz -out B7-295.reads.dat -st 454
------------------------------------------------------------------------------
MosaikBuild 2.1.73 2012-11-08
Michael Stromberg & Wan-Ping Lee Marth Lab, Boston College Biology Department
------------------------------------------------------------------------------

- setting read group ID to: ZKON5B26EGE
- setting sample name to: unknown
- setting sequencing technology to: 454

- parsing FASTQ file:
reads: 2,281,414 \ERROR: The number of qualities (45) do not match the number of bases (385) in 9IKNG:01351:01857.

Is everybody using newbler ? do you find the same problems of data inconsistency on the Ion Torrent fastq or bam/sam converted formats ?

Keep in touch and thanks in advance !

Alessandro

Hi, for ion torrent data, and de-novo assemblies - CLCworkbench, Newbler, Ray, Mira and CAP3 are very good assemblers. I have tried them all and they work very well.

Now for mapping assemblies against a reference - you can use CLCworkbench, Newbler and MIRA. Mira produce one consensus sequence concerning the reference assembly.

Hope this helps.

[aguffanti]

Thanks for the links, this is what I was looking for !

I will also experiment with the Ion Torrent Virtualized software

Yes the Mosaik Aligner output I sent was from a failed run (incomplete transfer of data)

This is what I get from C11-278 dataset (which, I uderstand, is DH10B). Does this make sense to you ? the real issue I found with these datasets (except tis one) is a quite relevant number of reads with 5 nt or less - can somebody already familiar with this technology comment on that ? I am a 5500 analyst, but former 454 (never mind the mates, these are fragments..)

Alignment statistics (mates):
================================================
# too short: 813 ( 0.0 %)
# failed hash: 1022 ( 0.0 %)
------------------------------------------------
# unaligned mates(X): 1835 ( 0.0 %)
# filtered out(F): 1313241 ( 19.5 %)
# uniquely aligned mates(U): 5045130 ( 74.8 %)
# multiply aligned mates(M): 382553 ( 5.7 %)
================================================
total aligned: 5427683 ( 80.5 %)
total: 6742759

MosaikAligner CPU time: 6379.710 s, wall time: 812.642 s

Thanks again to everybody and keep in touch

Alessandro

[IonTorrent]

Hi Alessandro,

TMAP is open source: https://github.com/iontorrent/TMAP

Also, if you were unable to access the link posted by jdilts, you can register for an account at the Ion Community to read further information on TMAP. You don't need to own an instrument.

Hi - I don't own the machine, so how do I get hold of TMAP ?

I am trying to evaluate the quality of the data BEFORE considering a purchase

Keep in touch

Alessandro

[jdilts]

ohhhh! I see. You are working on data from an Ion Torrent machine that you are no where near.

In that case I would have them load the genome as a reference in the machine and re-run the analysis.

If you can't do that, I would use MIRA.

I've never used Mosaik, but your results don't sound convincing. We have 9 PGMs total. They are cheap and work fantastic.

[aguffanti]

Re: assembly

Hi - as I said, I don't own a PGM, I am just experimenting in silico ;-)

However I finally suceeded in aligning at least the DH10B dataset to its genome with Mosaik - all the others seem to have an enormous number of reads nbelow 5 nt, which is quite bizarre..

Keep in touch

A

[jdilts]

TMAP comes with the PGM (You won't need to load anything) During the alignment phase of the analysis TMAP aligns the reads to a reference that you loaded automatically.

Just be sure you load a reference sequence (fasta) and use it when you plan your run.

http://ioncommunity.lifetechnologies.com/docs/DOC-1856

[aguffanti]

Re:

Hi - I don't own the machine, so how do I get hold of TMAP ?

I am trying to evaluate the quality of the data BEFORE considering a purchase

Keep in touch

Alessandro

[jdilts]

IonTor data: denovo w/MIRA, reference w/the ion community's built in assembler

Hi afuffanti,

I've used IonTor to do a denovo w/MIRA and I've also used a reference w/the ion community's built in aligner TMAP. Both were successful.

What are you trying to do?

[aguffanti]

Referenced assembly with Ion Torrent - Mosaik from fastq files

Hi - has anybody tried referenced or de novo assembly from the .sam (or .fastq) files and ion Torrent datasets ? I downloaded the following data sets from the ion Community:

B7-143
B7-295
C19-543

And I do have .bam (.sam) and .fastq files

Velvet de novo assembly technically worked, but left me with many thousands of small contigs, so it is useless

(*) Velvet

All trials of referenced assembly with the Columbus extension to Velvet and Mosaik Assembler (using sorted sam files) and Mosaik Assembler (MosaikBuild) failed, apparently for serious inconsistencies on the data set or data format incompatibility:
[0.000000] Reading FastA file NC_010473.C19-543.genome.fasta;
[59.568244] 1 sequences found
[59.568247] Done
[59.568619] Reading SAM file C19-543.sorted.sam
[355.404283] 6906611 reads found.
[355.404285] Done
[355.404286] Reference mapping counters
[355.404287] Name Read mappings
[355.404288] gi|170079663|ref|NC_010473.1| 19688393
[356.782277] Reading read set file C19-543/Sequences;
[363.732809] 6906612 sequences found
[363.733517] Read 1 of length 32794, longer than limit 32767
[363.733519] You should modify recompile with the LONGSEQUENCES option (cf. manual)

(*) MosaikBuild

MosaikBuild -q B7-295.fastq.gz -out B7-295.reads.dat -st 454
------------------------------------------------------------------------------
MosaikBuild 2.1.73 2012-11-08
Michael Stromberg & Wan-Ping Lee Marth Lab, Boston College Biology Department
------------------------------------------------------------------------------

- setting read group ID to: ZKON5B26EGE
- setting sample name to: unknown
- setting sequencing technology to: 454

- parsing FASTQ file:
reads: 2,281,414 \ERROR: The number of qualities (45) do not match the number of bases (385) in 9IKNG:01351:01857.

Is everybody using newbler ? do you find the same problems of data inconsistency on the Ion Torrent fastq or bam/sam converted formats ?

Keep in touch and thanks in advance !

Alessandro

[DNASTAR]

Hi All,

DNASTAR software fully supports reference-guided assemblies (as well as de novo) for Ion Torrent data. If you check out the Ion Torrent page on our website, you can see videos of many of the Ion Torrent project types we handle (Ion AmpliSeq Cancer Panel, Paired-End Assembly with a Reference, etc.), as well as benchmarks and other resources.

Also, feel free to download a fully-functional free trial of Lasergene Genomics Suite to try it out for yourself.

If you have questions, just give us a call or send us an email.
866-511-5090
[email protected]

Thanks,
Anne

[byou678]

Thanks for your info and advice.

Quick start for clcbio is to check their docs.
Their support page is at: http://www.clcbio.com/index.php?id=615 It has links to FAQ, tutorials, screencasts, etc.
Open up the app, see what it looks like, see how much is intuitive. There are many many more features than any one sequencing lab or bioinf group will use, so my usual way is to investigate the app, then check docs, then investigate, check docs, ask forum, investigate, repeat.

This clc-specific topic (if continued) is probably better as new thread.

-B-

[BenjaminL]

Quick start for clcbio is to check their docs.
Their support page is at: http://www.clcbio.com/index.php?id=615 It has links to FAQ, tutorials, screencasts, etc.
Open up the app, see what it looks like, see how much is intuitive. There are many many more features than any one sequencing lab or bioinf group will use, so my usual way is to investigate the app, then check docs, then investigate, check docs, ask forum, investigate, repeat.

This clc-specific topic (if continued) is probably better as new thread.

-B-

[slm1816]

I am also curious about the CLC software....I'm not exactly sure in how to use it.

[jonathanjacobs]

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?

@Ronan: We use GALAXY and CLCbio Genome Workbench in our shop and have a MiSeq and IonTorrent in house. we're mainly doing microbial and viral resequencing, but it seems as though de novo assembly keeps creeping in as well. In any case - CLCbio is --extremely-- fast (minutes) to do a 30x coverage of a 5MB genome for read mapping. The added benefit is that is also does true hybrid assembly of both PE and single read NGS data from both MiSeq and PGM at the same time. The quality/accuracy has also been as good, if not better, than some of the open source solutions we are running as well (with GALAXY). It's pricey (~$5K/license) - but the time savings in both setup and running is worth it. Don't get me wrong though - GALAXY is also very very good, but it took a while for me to get some of the tools we're using to install "right" to work with GALAXY.

POSTEDIT: The original post mentioed "reference assembly" - perhaps I've crossed some wires. I thinking "read mapping." For de novo assembly - CLCbio is also very fast and accurate. We routinely get down to the sub-100 contigs with single Ion318 or MiSeq PE runs for a 5MB genome. (N50 is on average around 190K)

[BenjaminL]

Length limiting is a Great idea jdilts.
One quick note on that quick perl script...

The concept for length checking is a good one, but this script fetches and measures each line as a read. If you are using a specific file type of the sequencer's reads the it will depend on the format.
e.g. fastq uses (at least) 4 lines for each section; including name, sequence, quality and one optional line. This is assuming that the sequence is all one line. A complete read may be longer than one section of the fastq as well.

To parse a specific file type (as opposed to one that has one line per read) then I recommend you either write a new function/method or use a prewritten library that does that. I know that bioperl and biopython have packages that read many file types, fastq being just one of them.

-Benjamin-