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Registration is now LIVE for our upcoming BroadE Workshop: Best Practices for Variant Calling with the GATK.

WHEN: Thursday, March 19 & Friday, March 20, 2015

10:00 AM - 5:00 PM (Lecture, March 19)
2:00PM - 5:00 PM (Optional Tutorial, March 20)

Auditorium (lecture)/Yellowstone (Tutorial)
415 Main Street
Cambridge, Massachusetts 02142

Registration Schedule:

Registration closes February 27 at 5:00 PM.
Notification of acceptance or wait list status sent by March 4.

REGISTER HERE

This workshop will focus on the core steps involved in calling variants with the Broad’s Genome Analysis Toolkit, using the “Best Practices” developed by the GATK team. The GATK development team and invited guests will give talks explaining the rationale, theory and real-life applications of the Best Practices. You will learn why each step is essential to the calling process, what are the key operations performed on the data at each step, and how to use the GATK tools to get the most accurate and reliable results out of your dataset.

An optional hands-on session will be available to select participants. In this session, the GATK team will help beginners work through interactive exercises and tutorials to learn how to use GATK and apply the Best Practices to real data.

Workshop attendees will gain broad insight into the rationale of the GATK Best Practices for variant discovery, as well as a solid understanding of how individual GATK tools work and how to apply them in practice. Novices to the GATK will come out of the workshop knowing enough to identify which questions they can use address using GATK tools, how to get started on designing their experiment and analytical workflow, and how to run the tools on their own computer. Existing GATK users will come out with a deeper understanding of how the GATK works under the hood and how to improve their results further, especially as regards the latest innovations.

We're going to be doing two back-to-back workshops in Edinburgh and Cambridge (the original, accept no substitutes) later this Spring, on April 20-21 and 23-24 respectively. The workshop program for both will be our typical one-day Best Practices lectures marathon followed by a half-day of lectures on supplemental topics (QC, non-humans, etc) and a half-day hands-on sessions for beginners to get their hands dirty with some real data.

These workshops are organized locally by the inviting institutions, University of Edinburgh and University of Cambridge, so please see the respective links for registration information.

Cheers to our hosts and we hope to see lots of you there!

bonus points to whoever gets the title reference -- and sings it in the correct tune

Warning: the following content may shock or distress our more sensitive users, as we discuss the cold-blooded elimination of some tools from the GATK.

Alright, now that I've got your attention (hopefully -- if not, what does it take?), here's the deal. We have got to a point where the GATK is a widely, even massively used toolkit (thanks to you, dear users). And it's pretty darn robust -- it's what the Broad's Genomic Platform uses in production to churn out exomes like there's no tomorrow. But it has technical limitations that are 1) a frequent source of pain on your end and 2) increasingly hampering development of new methods on our end.

The good news is that we have a plan for addressing (read: blasting away) these limitations. But part of this plan will involve streamlining GATK by getting rid of tools that are not useful or are inferior to alternative tools from other packages that we're not trying to compete with (e.g. Picard tools).

Some tools that are safe from elimination: all the tools used in the Best Practices, and a couple of utilities that we use a lot ourselves. But everything else is up for review -- and that's where you come in: we need your input to decide what to keep, what to throw away, and what to consider rewriting from scratch (yep, this is an option).

This link will take you to a SurveyMonkey page that lists the tools currently on the chopping block:

https://www.surveymonkey.com/s/RN8DR3D

Act now to save your favorite non-BP tools! Or help us get rid of the crud. Whichever way you want to look at it, we appreciate your feedback!

We all know how HaplotypeCaller analyses can take a long time. IBM is now providing a native implementation of the PairHMM algorithm that leverages the new hardware available in their POWER8 systems. This optimization currently work on the following systems: Ubuntu14 and RHEL7 with POWER8.

To take advantage of this optimization, you need to do the following:

• set your java library path to the location of libVectorLoglessPairHMM.so using -Djava.library.path
• opt-in by adding the following flag to your GATK command: -pairHMM VECTOR_LOGLESS_CACHING

Here is an example for running on a P8 system with Ubuntu:

java -Xmx32g -Djava.library.path=/path/to/PairHMM_P8_Ubuntu -jar $GATK_PATH/GenomeAnalysisTK.jar \ -T HaplotypeCaller \ -R$REFERENCE -I $INPUT_BAM --dbsnp$SNP_VCF \
-stand_emit_conf 10 -stand_call_conf 50 \
--pair_hmm_implementation VECTOR_LOGLESS_CACHING \
-o OUTPUT_VCF  You can expect a speedup in the range of 1-1.7x depending on the hardware, OS and test cases. If you have any questions or issues (aside from downloading the file), please contact Yinhue Cheng at IBM (ycheng@us.ibm.com). Consider this a public service announcement, since most GATK users probably also use Picard tools routinely. The recently released version 1.124 of the Picard tools includes many lovely improvements, bug fixes and even a few new tools (see release notes for full details) -- but you'll want to pay attention to one major change in particular. From this version onward, the Picard release will contain a single JAR file containing all the tools, instead of one JAR file per tool as it was before. This means that when you invoke a Picar tool, you'll invoke a single JAR, then specify which tool (which they call CLP for Command Line Program) you want to run. This should feel completely familiar if you already use GATK regularly, but it does mean you'll need to update any scripts that use Picard tools to the new paradigm. Other than that, there's no change of syntax; Picard will still use e.g. I=input.bam where GATK would use -I input.bam. We will need to update some of our own documentation accordingly over the near future; please bear with us as we go through this process, and let us know by commenting in this thread if you find any docs that have yet to be updated. Another season, another GATK release. Personally, Fall is my favorite season, and while I don’t want to play favorites with versions (though unlike with children, you’re allowed to say that the most recent one is the best --and you can tell I was a youngest child) this one is pretty special to me. Because -ploidy! Yeah, that’s really all I need to say about that. I was a microbiologist once. And I expect many plant people will be happy too. Other cool stuff detailed below includes: full functionality for the genotype refinement workflow tools; physical phasing and appropriate handling of dangly bits by HaplotypeCaller (must… resist… jokes…); a wealth of new documentation for variant annotations; and a slew of bug fixes that I won’t go over but are listed in the release notes. ### Genotype refinement workflow with all the trimmings As announced earlier this week, we recently developed a workflow for refining genotype calls, intended for researchers who need highly accurate genotype information as well as preliminary identification of possible de novo mutations (see the documentation for details). Although all the tools involved were already available in GATK 3.2, some functionalities were not, so we’re very happy to finally make all of them available in this new version. Plus, we like the new StrandOddsRatio annotation (which sort of replaces FisherStrand for estimating strand bias) so much that we made it a standard one, and it now gets annotated by default. ### Non-diploids, rejoice! This is also a feature that was announced a little while ago, but until now was only fully available in the nightly builds, which are technically unsupported unless we tell you to use them to get past a bad bug. In this new release, both HaplotypeCaller and GenotypeGVCFs are able to deal with non-diploid organisms (whether haploid or exotically polyploid). In the case of HaplotypeCaller, you need to specify the ploidy of your non-diploid sample with the -ploidy argument. HC can only deal with one ploidy at a time, so if you want to process different chromosomes with different ploidies (e.g. to call X and Y in males) you need to run them separately. On the bright side, you can combine the resulting files afterward. In particular, if you’re running the -ERC GVCF workflow, you’ll find that both CombineGVCFs and GenotypeGVCFs are able to handle mixed ploidies (between locations and between samples). Both tools are able to correctly work out the ploidy of any given sample at a given site based on the composition of the GT field, so they don’t require you to specify the -ploidy argument. ### HaplotypeCaller gets physical You know how HC performs a complete reassembly of reads in an ActiveRegion? (If you don’t, go read this now. Go on, we’ll wait for you.) Well, this involves building an assembly graph, of course (of course!), and it produces a list of haplotypes. Fast-forward a couple of steps, and you end up with a list of variants. That’s great, but until now, those variants were unphased, meaning the HC didn’t give you any information about whether any two variants’ alleles were on the same haplotype (meaning, on the same physical piece of DNA) or not. For example, you’d want to know whether you had this: or this: But HC wouldn’t tell you which it was in its output. Which was a shame, because the HC sees that information! It took a little tweaking to get it to talk, but now it emits physical phasing by default in its GVCF output (both banded GVCF and BP_RESOLUTION). In a nutshell, phased records will look like this: 1 1372243 . T <NON_REF> . . END=1372267 <snip> <snip> 1 1372268 . G A,<NON_REF> . . <snip> GT:AD:DP:GQ:PGT:PID:PL:SB 0/1:30,40,0:70:99:0|1:1372268_G_A:<snip> 1 1372269 . G T,<NON_REF> . . <snip> GT:AD:DP:GQ:PGT:PID:PL:SB 0/1:30,41,0:71:99:0|1:1372268_G_A:<snip> 1 1372270 . C <NON_REF> . . END=1372299 <snip> <snip>  You see that the phasing info is encoded in two new sample-level annotations, PID (for phase identifier) and PGT (phased genotype). More than two variants can be phased in a group with the same PID, and that can include mixed types of variants (e.g. SNPs and indels). The one big caveat related to the physical phasing output by HC in GVCFs is that, like the GVCF itself, it is not intended to be used directly for analysis! You must run your GVCFs through GenotypeGVCFs in order to get the finalized, properly formatted, ready-for-analysis calls. ### Heads or tails Speaking of HaplotypeCaller getting more helpful all the time, here’s some more of that. This still has to do with the graph assembly, and specifically, with how HC handles the bits at the edges of the graph, which are called dangling heads and dangling tails. Without going too far into the details, let’s just say that sometimes you have a variant that’s near the edge of a covered region, and due to technical reasons (cough kmer size cough) the end of the variant path can’t be tied back into the reference path, so it just dangles there (like, say, Florida) and gets trimmed off in the next step (rising ocean levels). And thus the variant is lost (boo). We originally started paying attention to this because it often happens at the edge of exons near splice junctions in RNAseq data, but it can also happen in DNA data. The solution was to give HC the ability to recover these cliff-dwelling variants by merging the dangling ends back into the graph using special logic tailored for those situations. If you have been using our RNAseq Best Practices, then you may recognize this as the logic invoked by the --recoverDanglingHeads argument. In the new version, the functionality has been improved further and is now enabled by default for all variant calling (so you no longer need to specify that argument for RNAseq analysis). The upshot is that sensitivity is improved, especially for RNAseq data but also for DNA. ### Variant annotations finally make sense Finally, I want to attract everyone’s attention to the Variant Annotations section of the Tool Documentation, which has just undergone a comprehensive overhaul. All annotations now have some kind of documentation outlining their general purpose, output, interpretation, caveats and some notes about how they’re calculated where applicable. Tell us what you think; we are feedback junkies. We're looking for a developer (software engineer or equivalent) to join our team on a part-time basis as a software engineering consultant. The mission is to take on tasks that are non-critical but still important, such as fixing bugs and implementing minor feature requests, usability improvements and so on in the GATK codebase. The idea is to take much of the maintenance burden off of the core development team so they can focus on developing new tools and methods. We already have one person employed in this capacity, and it's working out very well. However there is quite a bit more to do than he can find time for, and therefore we'd like to hire a second consultant to pick up the extra work in parallel. (We were hoping to just clone him but ran into some difficulties getting IRB approval.) This position does not require expert knowledge of GATK, but familiarity with the GATK tools is a big plus. The main language is Java, with a small side of R and Scala. We also have a growing corpus of C++ code that is not yet in the scope of this position, but may move into scope some months down the line. Note that this opportunity is amenable to remote work so you don't need to be local to the Boston area, but it is limited to US residents with valid work authorization (no H1B sponsorship possible, sorry). Drop us a line in the comments below or private-message me (@Geraldine_VdAuwera) to discuss details. Here is the official announcement for the upcoming workshop in Philadelphia. Registration is not necessary for the lecture sessions, but it is required for the hands-on sessions (see link further below). We look forward to seeing you there! The Center for Genetics and Complex Traits (CGACT) and the Institute for Biomedical Informatics (IBI) of the University of Pennsylvania Perelman School of Medicine announce a Workshop on Variants Discovery in Next Generation Sequence Data on September 18 and 19, 2014. This workshop will focus on the core steps involved in calling variants with the Broad Institute¹s Genome Analysis Toolkit (GATK), using the "Best Practices" developed by the GATK team, and will be presented by Dr. Geraldine Van der Auwera of the Broad Institute and other instructors from the GATK team. Participants will learn why each step is essential to the calling process, what are the key operations performed on the data at each step, and how to use the GATK tools to get the most accurate and reliable results out of their dataset. The workshop will take place over two consecutive days (September 18 and 19, 2014). In the morning lecture sessions, attendees will learn the rationale, theory, and real-life applications of GATK Best Practices for variant discovery in high-throughput DNA sequencing data, including recommendations for additional experimental designs and datatypes such as RNAseq. In the afternoon hands-on sessions, attendees will learn to interact with the GATK tools and apply them effectively through interactive exercises and tutorials. The morning lecture sessions will take place on Thursday, September 18, from 9:00 am to 12:30 pm, and Friday, September 19, from 9:00 am to 11:30 am, in the Dunlop Auditorium of Stemmler Hall, University of Pennsylvania, 3450 Hamilton Walk, Philadelphia, PA 19104. Both morning sessions are open to all participants and registration is not required. The afternoon hands-on sessions will take place on Thursday, September 18, from 2:00 pm to 5:30 pm, and Friday, September 19, from 1:00 pm to 4:30 pm. The September 18 hands-on session is aimed mainly at beginners (though familiarity with the command line environment is expected). The September 19 hands-on session is aimed at more advanced users who are already familiar with the basic GATK functions. Attendance to the hands-on sessions is limited to 20 participants each day, and precedence will be given to members of the University of Pennsylvania or its affiliated hospitals and research institutes (HUP, CHOP, Monell, Wistar, etc.). Registration for the hands-on sessions is mandatory and open through Friday, August 29th at http://ibi.upenn.edu/?p=996. Better late than never (right?), here are the version highlights for GATK 3.2. Overall, this release is essentially a collection of bug fixes and incremental improvements that we wanted to push out to not keep folks waiting while we're working on the next big features. Most of the bug fixes are related to the HaplotypeCaller and its "reference confidence model" mode (which you may know as -ERC GVCF). But there are also a few noteworthy improvements/changes in other tools which I'll go over below. ### Working out the kinks in the "reference confidence model" workflow The "reference confidence model" workflow, which I hope you have heard of by now, is that awesome new workflow we released in March 2014, which was the core feature of the GATK 3.0 version. It solves the N+1 problem and allows you to perform joint variant analysis on ridiculously large cohorts without having to enslave the entire human race and turning people into batteries to power a planet-sized computing cluster. More on that later (omg we're writing a paper on it, finally!). You can read the full list of improvements we've made to the tools involved in the workflow (mainly HaplotypeCaller and Genotype GVCFs) in Eric's (unusually detailed) Release Notes for this version. The ones you are most likely to care about are that the "missing PLs" bug is fixed, GenotypeGVCFs now accepts arguments that allow it to emulate the HC's genotyping capabilities more closely (such as --includeNonVariantSites), the AB annotation is fully functional, reference DPs are no longer dropped, and CatVariants now accepts lists of VCFs as input. OK, so that last one is not really specific to the reference model pipeline, but that's where it really comes in handy (imagine generating a command line with thousands of VCF filenames -- it's not pretty). ### HaplotypeCaller now emits post-realignment coverage metrics The coverage metrics (DP and AD) reported by HaplotypeCaller are now those calculated after the HC's reassembly step, based on the reads having been realigned to the most likely haplotypes. So the metrics you see in the variant record should match what you see if you use the -bamout option and visualize the reassembled ActiveRegion in a genome browser such as IGV. Note that if any of this is not making sense to you, say so in the comments and we'll point you to the new HaplotypeCaller documentation! Or, you know, look for it in the Guide. ### R you up to date on your libraries? We updated the plotting scripts used by BQSR and VQSR to use the latest version of ggplot2, to get rid of some deprecated function issues. If your Rscripts are suddenly failing, you'll need to update your R libraries. ### A sincere apology to GATK-based tool developers We're sorry for making you jump through all these hoops recently. As if the switch to Maven wasn't enough, we have now completed a massive reorganization/renaming of the codebase that will probably cause you some headaches when you port your tools to the newest version. But we promise this is the last big wave, and ultimately this will make your life easier once we get the GATK core framework to be a proper maven artifact. In a nutshell, the base name of the codebase has changed from sting to gatk (which hopefully makes more sense), and the most common effect is that sting.gatk classpath segments are now gatk.tools. This, by the way, is why we had a bunch of broken documentation links; most of these have been fixed (yay symlinks) but there may be a few broken URLs remaining. If you see something, say something, and we'll fix it. We discovered today that we made an error in the documentation article that describes the RNAseq Best Practices workflow. The error is not critical but is likely to cause an increased rate of False Positive calls in your dataset. The error was made in the description of the "Split & Trim" pre-processing step. We originally wrote that you need to reassign mapping qualities to 60 using the ReassignMappingQuality read filter. However, this causes all MAPQs in the file to be reassigned to 60, whereas what you want to do is reassign MAPQs only for good alignments which STAR identifies with MAPQ 255. This is done with a different read filter, called ReassignOneMappingQuality. The correct command is therefore: java -jar GenomeAnalysisTK.jar -T SplitNCigarReads -R ref.fasta -I dedupped.bam -o split.bam -rf ReassignOneMappingQuality -RMQF 255 -RMQT 60 -U ALLOW_N_CIGAR_READS  In our hands we see a bump in the rate of FP calls from 4% to 8% when the wrong filter is used. We don't see any significant amount of false negatives (lost true positives) with the bad command, although we do see a few more true positives show up in the results of the bad command. So basically the effect is to excessively increase sensitivity, at the expense of specificity, because poorly mapped reads are taken into account with a "good" mapping quality, where they would normally be discarded. This effect will be stronger in datasets with lower overall quality, so your results may vary. Let us know if you observe any really dramatic effects, but we don't expect that to happen. To be clear, we do recommend re-processing your data if you can, but if that is not an option, keep in mind how this affects the rate of false positive discovery in your data. We apologize for this error (which has now been corrected in the documentation) and for the inconvenience it may cause you. Calling all Belgians! (and immediate neighbors) In case you didn't hear of this through your local institutions, I'm excited to announce that we are doing a GATK workshop in Belgium in two weeks (June 24-26 to be precise). The workshop, which is open and free to the scientific community, will be held at the Royal Institute of Natural Sciences in Brussels. This is SUPER EXCITING to me because as a small child I spent many hours drooling in front of the Institute Museum's stunningly beautiful Iguanodons, likely leaving grubby handprints all over the glass cases, to the shame and annoyance of my parents. I also happen to have attended the Lycee Emile Jacqmain which is located in the same park, right next to the Museum (also within a stone's throw of the more recently added European Parliament) so for me this is a real trip into the past. Complete with dinosaurs! That said, I expect you may find this workshop exciting for very different reasons, such as learning how the GATK can empower your research and hearing about the latest cutting-edge developments that you can expect for version 3.2. See this website or the attached flyer for practical details (but note that the exact daily program may be slightly different than announced due to the latest changes in GATK) and be sure to register (it's required for admission!) by emailing cvangestel at naturalsciences.be with your name and affiliation. Please note that the hands-on sessions (to be held on the third day) are already filled to capacity. The tutorial materials will be available on our website in the days following the workshop. In a nutshell: if you're using a version of GATK older than 2.7, you need to request a key to disable Phone Home (if you don't already have one). See below for a full explanation. As you may know, the GATK includes a reporting mechanism called Phone Home that sends us runtime statistics about usage and bugs. These statistics (which you can read more about here) help us make development decisions, e.g. prioritize bug fixes and new features, as well as track adoption of new versions and tools. The system uses an AWS cloud service as a data repository, called a "bucket", which GATK accesses using an encryption key. We currently have two active AWS keys for the Phone Home bucket. GATK versions 2.6 and older use one AWS key, and versions 2.7 and later use another AWS key. For practical reasons, we need to deactivate the old AWS key, which means that GATK jobs run using a version older than 2.7 may end with a Phone Home failure unless the system is deactivated. To be clear, the GATK analysis itself will complete successfully, but the GATK may exit with a fail code. This may cause issues in pipelines and potentially fill up error logs. The best way to avoid these problems is to upgrade to the latest version of GATK, which you should seriously consider anyway in order to get the best possible results out of your analysis. However, if you are unable to upgrade to a recent version, we recommend that you disable the Phone Home system. To do so, you will need to follow these two simple steps: 1. Request a GATK key using this online form. In the "justification" field, please write "AWS key deprecation", indicate which version of GATK you are using, and if possible let us know why you are unable to upgrade to a recent version. You can expect to receive your key within 1 business day. 2. Once you have the key, apply it to all GATK jobs by adding -et NO_ET -K your.key (where your.key is the path to the key file you obtained from us) to every GATK command line. We expect this workaround will suppress any issues EXCEPT in versions prior to 1.5, in which the Phone Home system cannot be deactivated. For versions 1.4 and older, there is nothing we can do, and you will have to either put up with the errors, or upgrade to a newer version (which you should really really do anyway!). Let us know in the comments if you have any trouble or questions. This may seem crazy considering we released the big 3.0 version not two weeks ago, but yes, we have a new version for you already! It's a bit of a special case because this release is all about the hardware-based optimizations we had previously announced. What we hadn't announced yet was that this is the fruit of a new collaboration with a team at Intel (which you can read more about here), so we were waiting for everyone to be ready for the big reveal. ### Intel inside GATK So basically, the story is that we've started collaborating with the Intel Bio Team to enable key parts of the GATK to run more efficiently on certain hardware configurations. For our first project together, we tackled the PairHMM algorithm, which is responsible for a large proportion of the runtime of HaplotypeCaller analyses. The resulting optimizations, which are the main feature in version 3.1, produce significant speedups for HaplotypeCaller runs on a wide range of hardware. We will continue working with Intel to further improve the performance of GATK tools that have historically been afflicted with performance issues and long runtimes (hello BQSR). As always, we hope these new features will make your life easier, and we welcome your feedback in the forum! ### In practice Note that these optimizations currently work on Linux systems only, and will not work on Mac or Windows operating systems. In the near future we will add support for Mac OS. We have no plans to add support for Windows since the GATK itself does not run on Windows. Please note also that to take advantage of these optimizations, you need to opt-in by adding the following flag to your GATK command: -pairHMM VECTOR_LOGLESS_CACHING. Here is a handy little table of the speedups you can expect depending on the hardware and operating system you are using. The configurations given here are the minimum requirements for benefiting from the expected speedup ranges shown in the third column. Keep in mind that these numbers are based on tests in controlled conditions; in the wild, your mileage may vary. Linux kernel version Architecture / Processor Expected speedup Instruction set Any 64-bit Linux Any x86 64-bit 1-1.5x Non-vector Linux 2.6 or newer Penryn (Core 2 or newer) 1.3-1.8x SSE 4.1 Linux 2.6.30 or newer SandyBridge (i3, i5, i7, Xeon E3, E5, E7 or newer) 2-2.5x AVX To find out exactly which processor is in your machine, you can run this command in the terminal:  cat /proc/cpuinfo | grep "model name"
model name  : Intel(R) Core(TM) i7-2600 CPU @ 3.40GHz
model name  : Intel(R) Core(TM) i7-2600 CPU @ 3.40GHz
model name  : Intel(R) Core(TM) i7-2600 CPU @ 3.40GHz
model name  : Intel(R) Core(TM) i7-2600 CPU @ 3.40GHz
model name  : Intel(R) Core(TM) i7-2600 CPU @ 3.40GHz
model name  : Intel(R) Core(TM) i7-2600 CPU @ 3.40GHz
model name  : Intel(R) Core(TM) i7-2600 CPU @ 3.40GHz
model name  : Intel(R) Core(TM) i7-2600 CPU @ 3.40GHz


In this example, the machine has 4 cores (8-threads), so you see the answer 8 times. With the model name (here i7-2600) you can look up your hardware's relevant capabilities in the Wikipedia page on vector extensions.

Alternatively, Intel has provided us with some links to lists of processors categorized by architecture, in which you can look up your hardware:

#### Penryn processors

• http://ark.intel.com/products/codename/26543/Penryn
• http://ark.intel.com/products/codename/24736/Wolfdale
• http://ark.intel.com/products/codename/26555/Harpertown
• http://ark.intel.com/products/codename/25006/Dunnington

#### Sandy Bridge processors

• http://ark.intel.com/products/codename/29900/Sandy-Bridge?wapkw=sandy+bridge+processors

Finally, a few notes to clarify some concepts regarding Linux kernels vs. distributions and processors vs. architectures:

• SandyBridge and Penryn are microarchitectures; essentially, these are sets of instructions built into the CPU. Core 2, core i3, i4, i7, Xeon e3, e5, e7 are the processors that will implement a specific architecture to make use of the relevant improvements (see table above).

• The Linux kernel has no connection with Linux distribution (e.g. Ubuntu, RedHat etc). Any distribution can use any kernel they want. There are "default kernels" shipped with each distribution, but that's beyond the scope of this article to cover (there are at least 300 Linux distributions out there). But you can always install whatever kernel version you want.

• The kernel version 2.6.30 was released in 2009, so we expect every sane person or IT out there to be using something better than this.

Okay, we realize the name's a bit of a mouthful, and we're willing to tweak it if anyone has any good ideas. But never mind that. It's difficult to overstate the importance of this new approach to joint variant discovery (but I'll give it a shot) so we're really stoked to finally be able to share the details of how it's is going to work in practice.

You're probably going to be surprised at how simple it is in practice (not that it was particularly easy to actually build, mind you). The gory details are in the new document here, but here's an overview of how it looks within the Best Practices workflow you all know and (hopefully) love:

The first surprise is that instead of calling variants on multiple samples, you now just run HaplotypeCaller on each sample individually. "Oh no," I hear you cry, "but the results were so much better when I called multiple samples together!". Well yeah, but it took forever. Bear with me for a minute.

The key here is that you run HaplotypeCaller in gVCF mode. This outputs a so-called genomic VCF, which contains a record of the genotype likelihoods and annotations for every single site in the genome (or exome), whether or not there is evidence of variation. This essentially boils down all the useful information that can be gleaned from the BAM files, and makes it unnecessary to refer back to the BAM in later steps.

So you repeat that for all your samples (which goes reasonably fast since per-sample calling is pretty tractable nowadays). Optionally, you can add in a step to combine gVCF files if you're working on a really large cohort. Then in the next step, you just run a separate genotyping tool on all the gVCFs (or combined gVCFs) together, which gives you the same output (raw SNPs and indel calls) that you would have got from one-step multisample calling.

See, that's the beauty of the new workflow. A lot less work (for the computer) for equivalent results. And the ability to process samples incrementally and perform joint discovery on cohort sizes that would have previously got you hauled off to the funny farm.

Let us know what you think!

We’re excited to introduce our Best Practices recommendations for calling variants on RNAseq data. These recommendations are based on our classic DNA-focused Best Practices, with some key differences in the early data processing steps, as well as in the calling step.

### Best Practices workflow for RNAseq

This workflow is intended to be run per-sample; joint calling on RNAseq is not supported yet, though that is on our roadmap.

Please see the new document here for full details about how to run this workflow in practice.

In brief, the key modifications made to the DNAseq Best Practices focus on handling splice junctions correctly, which involves specific mapping and pre-processing procedures, as well as some new functionality in the HaplotypeCaller.

Now, before you try to run this on your data, there are a few important caveats that you need to keep in mind.

Please keep in mind that our DNA-focused Best Practices were developed over several years of thorough experimentation, and are continuously updated as new observations come to light and the analysis methods improve. We have only been working with RNAseq for a few months, so there are many aspects that we still need to examine in more detail before we can be fully confident that we are doing the best possible thing.

For one thing, these recommendations are based on high quality RNA-seq data (30 million 75bp paired-end reads produced on Illumina HiSeq). Other types of data might need slightly different processing. In addition, we have currently worked only on data from one tissue from one individual. Once we’ve had the opportunity to get more experience with different types (and larger amounts) of data, we will update these recommendations to be more comprehensive.

Finally, we know that the current recommended pipeline is producing both false positives (wrong variant calls) and false negatives (missed variants) errors. While some of those errors are inevitable in any pipeline, others are errors that we can and will address in future versions of the pipeline. A few examples of such errors are given in this article as well as our ideas for fixing them in the future.

We will be improving these recommendations progressively as we go, and we hope that the research community will help us by providing feedback of their experiences applying our recommendations to their data. We look forward to hearing your thoughts and observations!

Previously, we covered the spirit of GATK 3.0 (what our intentions are for this new release, and what we’re hoping to achieve). Let’s now have a look at the top three features you can look forward to in 3.0, in no particular order:

1. Optimized PairHMM algorithm to make GATK run faster
2. Single-sample pipeline for joint variant discovery
3. Best practices for calling variants on RNAseq data

### 1. Optimized PairHMM algorithm to make HaplotypeCaller faster

At this point everyone knows that the HaplotypeCaller is fabulous (you know this, right?) but beyond a certain number of samples that you’re trying to call jointly, it just grinds to a crawl, and any further movement is on the scale of continental drift. Obviously this is a major obstacle if you’re trying to do any kind of work at scale beyond a handful of samples, and that’s why it hasn’t been used in recent large-cohort projects despite showing best-in-class performance in terms of discovery power.

The major culprit in this case is the PairHMM algorithm, which takes up the lion’s share of HC runtime. With the help of external collaborators (to be credited in a follow-up post) we rewrote the code of the PairHMM to make it orders of magnitude faster, especially on specialized hardware like GPU and FPGA chips (but you’ll still see a speedup on “regular” hardware).

We plan to follow up on this by doing similar optimizations on the other “slowpoke” algorithms that are responsible for long runtimes in GATK tools.

### 2. Single-sample pipeline for joint variant discovery

Some problems in variant calling can’t be solved by Daft Punk hardware upgrades (better faster stronger) alone. Beyond the question of speed, a major issue with multi-sample variant discovery is that you have to wait until all the samples are available to call variants on them. Then, if later you want to add some more samples to your cohort, you have to re-call all of them together, old and new. This, also known as the “N+1 problem”, is a huge pain in the anatomy.

The underlying idea of the “single-sample pipeline for joint variant discovery” is to decouple the two steps in the variant calling process: identifying evidence of variation, and interpreting the evidence. Only the second step needs to be done jointly on all samples, while the first step can be done just as well (and a heck of a lot faster) on one sample at a time.

The new pipeline allows us to process each sample as it comes off the sequencing machine, up to the first step of variant calling. Cumulatively, this will produce a database of per-sample, per-site allele frequencies. Then it’s just a matter of running a joint analysis on the database, which can be done incrementally each time a new sample is added or at certain intervals or timepoints, depending on the research needs, because this step runs quickly and cheaply.

We’ll go into the details of exactly how this works in a follow-up post. For now, the take-home message is that it’s a “single-sample pipeline” because you do the heavy-lifting per-sample (and just once, ever), but you are empowered to perform “joint discovery” because you interpret the evidence from each sample in light of what you see in all the other samples, and you can do this at any point in the project timeline.

### 3. Best practices for calling variants on RNAseq

Our Best Practices recommendations for calling variants on DNA sequence data have proved to be wildly popular with the scientific community, presumably because it takes a lot of the guesswork out of running GATK, and provides a large degree of reproducibility.

Now, we’re excited to introduce our Best Practices recommendations for calling variants on RNAseq data. These recommendations are based on our classic DNA-focused Best Practices, with some key differences the early data processing steps, as well as in the calling step. We do not yet have RNAseq-specific recommendations for variant filtering/recalibration, but will be developing those in the coming weeks.

We’ll go into the details of the RNAseq Best Practices in a follow-up post, but in a nutshell, these are the key differences: use STAR for alignment, add an exon splitting and cleanup step, and tell the variant caller to take the splits into account. The latter involves some new code added to the variant callers; it is available to both HaplotypeCaller and UnifiedGenotyper, but UG is currently missing a whole lot of indels, so we do recommend using only HC in the immediate future.

Keep in mind that our DNA-focused Best Practices were developed over several years of thorough experimentation, and are continuously updated as new observations come to light and the analysis methods improve. We have only been working with RNAseq for a few months, so there are many aspects that we still need to examine in more detail before we can be fully confident that we are doing the best possible thing. We will be improving these recommendations progressively as we go, and we hope that the researcher community will help us by providing feedback of their experiences applying our recommendations to their data.

Yep, you read that right, the next release of GATK is going to be the big Three-Oh!

You may have noticed that the 2.8 release was really slim. We explained in the release notes, perhaps a tad defensively, that it was because we’d been working on some ambitious new features that just weren’t ready for prime time. And that was true. Now we’ve got a couple of shiny new toys to show for it that we think you’re really going to like.

But GATK 3.0 is not really about the new features (otherwise we’d just call it 2.9). It’s about a shift in the way we approach the problems that we want to solve -- and to some extent, a shift in the scope of problems we choose to tackle.

We’ll explain what this entails in much more detail in a series of blog posts over the next few days, but let me reassure you right now on one very important point: there is nothing in the upcoming release that will disrupt your existing workflows. What it will do is offer you new paths for discovery that we believe will empower research on a scale that has previously not been possible.

And lest you think this is all just vaporware, here’s a sample of what we have in hand right now: variant calling on RNA-Seq, and a multisample variant discovery workflow liberated from the shackles of time and scaling issues.

Stay tuned for details!

The presentation videos for:

• Day 1 (Best Practices for Variant Calling with GATK)
• Day 2 (Building Analysis Pipelines with Queue)

Better late than never, here are the highlights of the most recent version release, GATK 2.8. This should be short and sweet because as releases go, 2.8 is light on new features, and is best described as a collection of bug fixes, which are all* dutifully listed in the corresponding release notes document. That said, two of the changes we've made deserve some additional explanation.

* Up to now (this release included) we have not listed updates/patches to Queue in the release notes, but will start doing so from the next version onward.

### VQSR & bad variants: no more guessing games

In the last release (2.7, for those of you keeping score at home) we trumpeted that the old -percentBad argument of VariantRecalibrator had been replaced by the shiny new -numBad argument, and that this was going to be awesome for all sorts of good reasons, improve stability and whatnot. Weeeeeeell it turned out that wasn't quite the case. It worked really well on the subset of analyses that we tested it on initially, but once we expanded to different datasets (and the complaints started rolling in on the forum) we realized that it actually made things worse in some cases because the default value was less appropriate than what -percentBad would have produced. This left people guessing as to what value would work for their particular dataset, with a great big range to choose from and very little useful information to assist in the choice.

So, long story short, we (and by "we" I mean Ryan) built in a new function that allows the VariantRecalibrator to determine for itself the amount of variants that is appropriate to use for the "bad" model depending on the data. So the short-lived -numBad argument is gone too, replaced by... nothing. No new argument to specify; just let the VariantRecalibrator do its thing.

Of course if you really want to, you can override the default behavior and tweak the internal thresholds. See the tool doc here; and remember that a good rule of thumb is that if you can't figure out which arguments are involved based on that doc, you probably shouldn't be messing with this advanced functionality.

### Reference calculation model

This is still a rather experimental feature, so we're still making changes as we go. The two big changes worth mentioning here are that you can now run this on reduced reads, and that we've changed the indexing routine to optimize the compression level. The latter shouldn't have any immediate impact on normal users, but it was necessary for a new feature project we've been working on behind the scenes (the single-sample-to-joint-discovery pipeline we have been alluding to in recent forum discussions). The reason we're mentioning it now is that if you use -ERC GVCF output, you'll need to specify a couple of new arguments as well (-variant_index_type LINEAR and -variant_index_parameter 128000, with those exact values). This useful little fact didn't quite make it into the documentation before we released, and not specifying them leads to an error message, so... there you go. No error message for you!

### What's up, doc?

That's all for tool changes. In addition to those, we have made a number of corrections in the tool documentation pages, updated the Best Practices (mostly layout, tiny bit of content update related to the VQSR -numBad deprecation) and made some minor changes to the website, e.g. updated the list of publications that cite the GATK and improved the Guide index somewhat (but that's still a work in progress).

Heads up, people: our generous overlords at the Broad Institute are giving us all time off from December 23 until January 1st (included). So we will effectively be off-duty starting tomorrow evening (Friday Dec 20) for the entire Christmas and New Year period, only to return on January 2nd. During that time, we will all be busy stuffing ourselves with food and enjoying the company of our loved ones, and we hope many of you will have the opportunity to do the same, regardless of your cultural affiliations (I for one will be raising a glass of mulled wine in honor of my Gaul ancestors and the winter solstice). For those of you who will be working, I'm afraid no-one from the GATK team will be around to answer forum questions (unless one of us really needs an excuse to get away from the in-laws) so I encourage you to try to answer each other's questions in the meantime. To all, good luck, happy holidays and/or our deepest sympathy, as applicable. See you next year!

We're very pleased to announce that we have finally finished our big rewrite of the Best Practices documentation. We hope that the new format, which you can find here, will prove more user-friendly, searchable and overall more helpful than the previous version.

We have a few more improvements in mind (e.g. a clickable image map of the workflow) and there may be a few bugs here and there to iron out. So please feel free to comment on this announcement and give us feedback, whether flattering or critical, so we can improve it to help you as much as possible.

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