Other memorable quotes
"Now you're telling me Martians with long stretches of repeats can't be sequenced?"
I wonder if there will be a video retort from the other camp.
Showing posts with label Ion Torrent. Show all posts
Showing posts with label Ion Torrent. Show all posts
Tuesday, 27 November 2012
I want to seq every martian for $1000 ..lol
This is pretty funny poke at the Ion Torrent (&Proton) vs MiSeq debate!
Other memorable quotes
I wonder if there will be a video retort from the other camp.
Other memorable quotes
I wonder if there will be a video retort from the other camp.
Tuesday, 15 May 2012
NATURE BIOTECHNOLOGY | Performance comparison of benchtop high-throughput sequencing platforms
NATURE BIOTECHNOLOGY | RESEARCH | ANALYSIS
Performance comparison of benchtop high-throughput sequencing platforms
- Nature Biotechnology
- 30,
- 434–439
- (2012)
- doi:10.1038/nbt.2198
Saturday, 18 February 2012
Oxford Nanopore megaton announcement: “Why do you need a machine?” – exclusive interview for this blog!
http://pathogenomics.bham.ac.uk/blog/2012/02/oxford-nanopore-megaton-announcement-why-do-you-need-a-machine-exclusive-interview-for-this-blog/
woke up this morning to see a whole bunch of excited tweets on Oxford Nanopore and I can totally understand why. This is the real democratization of DNA sequencing. Move over benchtop / desktop sequencers for 'laptop sequencers'!
Hmmm or a cluster of sequencers, on your compute cluster ... !
Hmmm or a cluster of sequencers, on your compute cluster ... !
Using USB powered sequencers, and a pipette to put in the dsDNA and you might have your sequence read to FASTQ directly to your laptop.
No known limit to read length.
4% seq error (the good thing is that the form of error is known and therefore correctable)
Do read the url above for more info, here's the excerpted executive summary for the impatient
Executive Summary
- Nanopore have announced a strand sequencing method, made possible by a heavily modified biological nanopore and an industrially-fabricated polymer
- DNA passes through the nanopore and tri-nucleotides in contact with the pore are detected through electrochemistry
- Demonstrated 2x50kb sense & anti-sense of same molecules (lambda phage) – no theoretical read length limit
- Can sequence direct from blood without need for sample preparation
- Two products announced:
- MinIon – USB disposable sequencer for ~ $900 has 512 nanopores – target 150mb/hour
- MinIon can run at 120-1000 bases/minute per pore for up to 6 hours
- GridIon – two versions of rack-mountable sequencer with 2000 nanopores (2nd half 2012), 8000 nanopores (2013)
- GridIons can be racked in parallel, 20 could do a whole human genome in 15 minutes
- Each GridIon can do "tens of gigabases" over 24 hours
- Both machines commercially available 2nd half 2012
- Sequencing can be paused, sample recovered, replaced, started again
- Accuracy is 96%, errors are deletions, error profile will improve through software
Check out Forbes interview with 454 / PGM inventor Jon Rothberg
"Rothberg noted that Ion Torrent’s new machine, the Proton, the company showed three completed human genomes yesterday at AGBT. More importantly, he had the machine – not a mock-up or a design – on the stage. “That’s where you need to be to ship mid-year,” he writes."
Over at Genomes Unzipped
Oxford Nanopore CTO Clive Brown related how sequencing library prep is as simple as diluting rabbit's blood with water. Now that is impressive!
This post is getting too long because I keep updating it.
Over at the BioITWorld, there's an interview with Clive Brown which cites other interesting info.
First of which is the opening paragraph which is amusing in the light of ONT's rivals comments
"Clive Brown, vice president of development and informatics for Oxford Nanopore Technologies (ONT), a.k.a “the most honest guy in all of next-gen sequencing,” as dubbed by The Genome Center's David Dooling, is hoping to catch lightning in a bottle again. "
Oxford Nanopore has not yet revealed details of its future platform, but in early 2009, published a lovely paper in Nature Nanotechnology showing that its alpha-hemolysin nanopores can discriminate between the four bases of DNA (not to mention a fifth, methyl C)
Directly get methylation information from your sequencing sans complicated sample prep? That has to be another selling point.
Not sure whether Nanopore is truly vaporware. However, gauging by the excitement over the blogosphere and the hit rates for the first to blog about it. I think Nanopore is upping the ante for the next IT sequencer.
maybe we can only survive 2 more AGBT like this and AGBT might fizzle out as new sequencing technologies fade as our computation advances trails behind the ability to generate more data.
Maybe you will see scientists start attending Big Data tech conferences or AGBT's main draw will fancy new software to assemble, align and make sense out of all the data being generated ...
This picture tells quite a story (Wordle constructed from 3,386 tweets and retweets tagged #AGBT with @s removed).
No prizes for guessing the winner ...
Friday, 17 February 2012
a tour of various bioinformatics functions in Avadis NGS
Not affliated with Avadis but this might be useful for you
We are hosting an online seminar series on the alignment and analysis of genomics data from “benchtop” sequencers, i.e. MiSeq and Ion Torrent. Our webinar panelists will give a tour of various bioinformatics functions in Avadis NGS that will enable researchers and clinicians to derive biological insights from their benchtop sequencing data.
Seminar #1: MiSeq Data Analysis
Avadis NGS 1.3 provides special support for analyzing data generated by MiSeq™ sequencers. In this webinar, we will describe how the data in a MiSeq generated “run folder” is automatically loaded into the Avadis NGS software during small RNA alignment and DNA variant analysis. This is especially helpful in processing the large number of files generated when the TruSeq™ Amplicon Kits are used. We will describe how to use the Quality Control steps in Avadis NGS to check if the amplicons have sufficient coverage in all the samples. Regions with unexpected coverages can easily be identified using the new region list clustering feature. Webinar attendees will learn how to use the “Find Significant SNPs” feature to quickly identify high-confidence SNPs present in a majority of the samples, rare variants, etc.
Seminar #2: Ion Torrent Data Analysis
Avadis NGS 1.3 includes a new aligner – COBWeb – that is fully capable of aligning the long, variable-length reads generated by Ion Torrent sequencers. In this webinar, we will show the pre-alignment QC plots and illustrate how they can be used to set appropriate alignment parameters for aligning Ion Torrent reads. For users who choose to import the BAM format files generated by the Ion Torrent Server, we will describe the steps needed for importing amplicon sequencing data into Avadis NGS. Users of the Ion AmpliSeq™ Cancer Panel will learn how to easily import the targeted mutation list and verify the genotype call at the mutation sites. We will also show the new “Find Significant SNPs” feature which helps quickly identify high-confidence SNPs present in a majority of the samples, rare variants, etc.
Free registration - http://www.avadis-ngs.com/webinar
We are hosting an online seminar series on the alignment and analysis of genomics data from “benchtop” sequencers, i.e. MiSeq and Ion Torrent. Our webinar panelists will give a tour of various bioinformatics functions in Avadis NGS that will enable researchers and clinicians to derive biological insights from their benchtop sequencing data.
Seminar #1: MiSeq Data Analysis
Avadis NGS 1.3 provides special support for analyzing data generated by MiSeq™ sequencers. In this webinar, we will describe how the data in a MiSeq generated “run folder” is automatically loaded into the Avadis NGS software during small RNA alignment and DNA variant analysis. This is especially helpful in processing the large number of files generated when the TruSeq™ Amplicon Kits are used. We will describe how to use the Quality Control steps in Avadis NGS to check if the amplicons have sufficient coverage in all the samples. Regions with unexpected coverages can easily be identified using the new region list clustering feature. Webinar attendees will learn how to use the “Find Significant SNPs” feature to quickly identify high-confidence SNPs present in a majority of the samples, rare variants, etc.
Seminar #2: Ion Torrent Data Analysis
Avadis NGS 1.3 includes a new aligner – COBWeb – that is fully capable of aligning the long, variable-length reads generated by Ion Torrent sequencers. In this webinar, we will show the pre-alignment QC plots and illustrate how they can be used to set appropriate alignment parameters for aligning Ion Torrent reads. For users who choose to import the BAM format files generated by the Ion Torrent Server, we will describe the steps needed for importing amplicon sequencing data into Avadis NGS. Users of the Ion AmpliSeq™ Cancer Panel will learn how to easily import the targeted mutation list and verify the genotype call at the mutation sites. We will also show the new “Find Significant SNPs” feature which helps quickly identify high-confidence SNPs present in a majority of the samples, rare variants, etc.
Free registration - http://www.avadis-ngs.com/webinar
Wednesday, 14 September 2011
Adding custom reference genome to Torrent Server manually - My Experience
Apologies! After digging in the Ion Community a little more, I think this is the updated link for V1.4 TS
Adding a New Genome Index
Created on: Jul 7, 2011 4:29 PM by ghartsell - Last Modified: Jul 11, 2011 1:48 PM by ghartsell
But the manually created reference index doesn't appear in the final dropdown menu when I try to do realignment (it does appear in the reference tab)
Don't really understand this line "As of release 1.1.0, only the "tmap-f1" index_type is supported."
as the index i created had the info.txt with tmap-f2
In anycase, if you don't mind fiddling with the web browser and you met with 'file deleted' or job started and you still do not have ur index . you can
restart ionJObServer
sudo /etc/init.d/ionJobServer restart
Adapted from the original doc here
Adding
a New Genome Index
As part of the standard analysis process reads are aligned to a genomic reference and the alignments and some summary statistics based on the alignments are included in the analysis report page. This HOWTO describes the process to add a new reference genome, something that will be necessary when a user starts to work with a new genome sequence.
The aligner used is named tmap and it comes pre-installed on the Torrent Server.
Prerequisites
Before we begin, you will need your reference sequence in a single file in fasta format and you will need command-line access to the Torrent Server. Please note that it must have Unix line endings and not Windows line endings. (they can be in .zip compressed format but i didn't test this)
Procedure
Select
a Short Form of Genome Name
The short form of genome name is the name that you would like the reference option to appear when initiating a run on the PGM™ instrument. There are some rules on how to define the short form of the genome name.
it should not match any of the existing references installed under the standard reference locationit should also be comprised solely of alphanumeric characters, underscore ("_") and period (".")
Index
Creation
The alignment package ( ion-alignment ) comes a wrapper script, build_genome_index.pl, that automates the TMAP index creation process. It requires four inputs:
single FASTA fileshort form of the genome name (see previous section)long form of the genome name (see next section for description)genome version (see next section for description)
The steps to create the index:
move or copy the FASTA file to the standard reference locationrun under the standard reference location
$ cd /results/referenceLibrary/tmap-f2/ $ build_genome_index.pl --fasta A_flavithermus.fasta -s A_flavithermus
-v "gi|212637849|ref|NC_011567.1"
-l "Anoxybacillus flavithermus WK1 chromosome complete genome" Copying A_flavithermus.fasta to A_flavithermus/A_flavithermus.fasta... ...copy complete Making tmap index... ...tmap index complete Making samtools index... ...samtools index complete
There should now be 10 files in the directory, including the original fasta file. The size of the files varies by genome - for the human genome (3,000,000,000 bases in length) the combined size of all index files, including the original fasta file itself, is just under 8Gb. For E. coli (4,600,000 bases in length) it is about 0.4Gb.
$ ls -1 /results/referenceLibrary/tmap-f1/e_coli/ A_flavithermus.fasta A_flavithermus.fasta.fai A_flavithermus.fasta.md5 A_flavithermus.fasta.tmap.anno A_flavithermus.fasta.tmap.bwt A_flavithermus.fasta.tmap.pac A_flavithermus.fasta.tmap.rbwt A_flavithermus.fasta.tmap.rpac A_flavithermus.fasta.tmap.rsa A_flavithermus.fasta.tmap.sa A_flavithermus.info.txt samtools.log tmap.log
Adding
the Genome to the PGM Drop-down Menu
For additional convenience it is also recommended (though not required) to add the genome to the list that is made available on the PGM as a drop-down menu - this can be very helpful in avoiding typos on the PGM.
updateref will crawl through the directory and grab the genome_shortname fields from all installed reference library of the version specified and overwrite reference_list.txt. When updateref is called without any command line argument, it will assume the default settings. For example, /results/PGM_config is the location of PGM configuration. The location is crucial because it needs to be under the same root directory to which PGM transfer the data. For example, if PGMs transfer data to a file server, which is mounted as /mnt/PGM_Data on Torrent server, an option -p /mnt/PGM_Data/PGM_config needs to be specified. updateref --help will list more options.
Default settings. PGM data are stored in /results.
$ sudo updateref List of library -> ampl_valid -> vibrio_fisch -> e_coli_k12 -> e_coli_dh10b -> rhodopalu
Customized environment. PGM data are stored in /mnt/PGM_Data.
$ sudo updateref -p /mnt/PGM_Data/PGM_config
Bash autocomplete is turned off by default in Torrent Server
Gah! Can't believe this feature is turned off in stock installations of Torrent Server. :)
Thankfully Ubuntu documentation is a plenty
http://embraceubuntu.com/2006/01/28/turn-on-bash-smart-completion/
To enable smart completion, edit your
Update: hmmm got bash completion to work for admin user but not the ionguest .. suspect is some bash issues :(
Update 2: yes the reason is because the default bash login shell is /bin/sh and not /bin/bash which you can change using the chsh command
tip courtesy of
http://linuxwave.blogspot.com/2009/03/changing-default-shell-in-ubuntu.html
Thankfully Ubuntu documentation is a plenty
http://embraceubuntu.com/2006/01/28/turn-on-bash-smart-completion/
To enable smart completion, edit your
/etc/bash.bashrc file. Uncomment the following lines, by removing the # in the beginning of the lines:
#if [ -f /etc/bash_completion ]; then
# . /etc/bash_completion
#fiUpdate: hmmm got bash completion to work for admin user but not the ionguest .. suspect is some bash issues :(
Update 2: yes the reason is because the default bash login shell is /bin/sh and not /bin/bash which you can change using the chsh command
tip courtesy of
http://linuxwave.blogspot.com/2009/03/changing-default-shell-in-ubuntu.html
Thursday, 18 August 2011
Tuesday, 2 August 2011
Nick Loman blogs PGM 316 1st Impressions
While eagerly waiting for our own PGM to be installed for 316 chip support, I came across Nick's review on their own 316 run. Which has pretty impressive output!
"Our first two runs of 316 chips yielded an impressive 251Mb and 209Mb respectively! Mean read length was about 110bp."
Other interesting factoids for the impatient
we've loaded the chips way higher than we are used to with the 314 – densities of 76-82%
This reflects a change to the protocol – when we were running 314 chips we were told to load fewer beads to get better coverage – and from our trials when we loaded at 41, 43 and 46% density on the 314 chip the 41% run did do best. The 314 chip has about 1.2m wells, so we were filling about 550k wells. About two-thirds of those wells were live spheres (meaning they have DNA on them) and out of those about two-thirds pass the quality filter – about 200k reads in all (~20Mb data).
The 316 chip has 6.3m wells and we're filling about 5m of these. A little under half are passing the quality filters, meaning we're getting about 2.25m reads.
Do hop on over to the original post to see FASTQC plots of the reads
Ion Torrent 316 First Impressions
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