The Genome Analysis Toolkit (GATK) is a set of programs developed by the broad institute with an extensive website. As mentioned in the final presentation, it has the ability to perform much of the analysis required for calling genomic variants as well as many many other things. Why you may ask yourself did this magical tool only appear on the final day of the class? GATK uses read mappers, read aligners, variant callers, and all the other things (or similar things) that you have been introduced to throughout the course so we have actually been going over what you needed to know in smaller more digestible chunks.
This tutorial is quite small and does not showcase but the smallest drop in a bucket of what GATK is capable of doing. This is because the broad itself has developed many many tutorials for all the different things GATK does and extensive forums are available if the tutorials are not enough to get you through what you are trying to do. Finally, as the makers of the software they have put out and maintain what they regard as the best way to use their product in the form of 'best practices'. If you are going to use GATK, its a real real real good idea to make sure you are following their best practices because that is a situation where people will raise a big eyebrow if you say you are going against the flow.
While GATK is great, one stop shops often are often not the best at everything they do, don't be afraid to use other programs. Particularly following what other researchers are doing in your field |
While you may think based on the overview that GATK is an obvious choice for a module on TACC, you may be surprised to learn that seemingly every other year TACC removes it as a module, and this is a bad year. On the plus side, it means that once we install it for you locally, the only issue will be if you need to update the version, and recent changes to GATK have made it much easier to work with.
# set up directories mkdir $WORK/src mkdir -p $HOME/local/bin # download file and extract it cd $WORK/src wget https://github.com/broadinstitute/gatk/releases/download/4.1.7.0/gatk-4.1.7.0.zip unzip gatk-4.1.7.0.zip # copy executables to somewhere already in your $PATH variable (remember we set this up on Monday in your .bashrc file) cp gatk-4.1.7.0/gatk $HOME/local/bin cp gatk-4.1.7.0/*.jar $HOME/local/bin # verify correctly installed cds gatk -help # if this does not output a large list of colored text, try the following command and if that does not output colored text get my attention gatk --list |
If you see 316 lines of a long scrolling output detailing some copyright information and a bunch of different commands everything is correctly loaded. While individual tools will require different options and the program itself takes many different options only 3 things are ALWAYS required:
| flag | Description |
|---|---|
| Tool name, what tool are you trying to use | |
| -R | Reference sequence file |
| -I | Input bam file |
Stealing a nice mnemonic devices from a GATK toturial (which is condensed below), these 3 arguments don't have to be in this order, but if you learn them in this order, you will be able to remember them if you TRI. Remember, specific tools will require additional arguments.
Rather than using sample data specifically for this tutorial, we will instead do a small tutorial based on our read mapping tutorial from day 2 of the course. Assuming you completed that tutorial you the following tutorial should work.
mkdir $SCRATCH/GVA_GATK cd $SCRATCH/GVA_GATK cp /scratch/01821/ded/GVA_bowtie2_mapping/bowtie2/SRR030257.sam . cp /scratch/01821/ded/GVA_bowtie2_mapping/NC_012967.1.fasta . |
Next you need to convert the .sam file to a .bam file.
samtools view -S -b SRR030257.sam > SRR030257.bam # Do you remember what tutorial we used this command in before? |
Using the following information we will use gatk the CountReads tool to count the number of reads in the SRR030257.bam file which was from the NC_012967.fasta reference file. Pay attention to the the words in bold and the table/discussion in the previous tutorial section and see if you can figure out how to do this on your own.
gatk CountReads -R NC_012967.1.fasta -I SRR030257.bam |
The following line tells us things did not go as intended.
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The text clearly tells us that a fasta index file is missing, and from the gatk link, we see that they are using the samtools faidx command to generate their indexes of fasta files.
samtools faidx NC_012967.1.fasta |
Now we repeat our CountReads command, and see that the results have indeed changed.
The following line tells us things still did not go as intended.
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That link contains a discussion about a .dict file that is required for fasta references that might normally be generated by GATK but as we did not do the read mapping inside of GATK we lack it. Based on the reading on the linked web page I came up with the following solution to create that dictionary file.
gatk CreateSequenceDictionary -R NC_012967.1.fasta -O NC_012967.1.dict |
Now if we retry our CountReads command we get very different output (note that yours will have subtle differences in things like the names of directories):
INFO: Failed to detect whether we are running on Google Compute Engine. 01:26:37.269 INFO CountReads - ------------------------------------------------------------ 01:26:37.269 INFO CountReads - The Genome Analysis Toolkit (GATK) v4.1.7.0 01:26:37.269 INFO CountReads - For support and documentation go to https://software.broadinstitute.org/gatk/ 01:26:37.269 INFO CountReads - Executing as ded@login2 on Linux v4.4.103-6.38-default amd64 01:26:37.269 INFO CountReads - Java runtime: OpenJDK 64-Bit Server VM v1.8.0_151-b12 01:26:37.269 INFO CountReads - Start Date/Time: June 24, 2020 1:26:37 AM CDT 01:26:37.269 INFO CountReads - ------------------------------------------------------------ 01:26:37.270 INFO CountReads - ------------------------------------------------------------ 01:26:37.270 INFO CountReads - HTSJDK Version: 2.21.2 01:26:37.270 INFO CountReads - Picard Version: 2.21.9 01:26:37.270 INFO CountReads - HTSJDK Defaults.COMPRESSION_LEVEL : 2 01:26:37.270 INFO CountReads - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false 01:26:37.270 INFO CountReads - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true 01:26:37.270 INFO CountReads - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false 01:26:37.270 INFO CountReads - Deflater: IntelDeflater 01:26:37.270 INFO CountReads - Inflater: IntelInflater 01:26:37.270 INFO CountReads - GCS max retries/reopens: 20 01:26:37.270 INFO CountReads - Requester pays: disabled 01:26:37.270 INFO CountReads - Initializing engine 01:26:37.589 INFO CountReads - Done initializing engine 01:26:37.589 INFO ProgressMeter - Starting traversal 01:26:37.589 INFO ProgressMeter - Current Locus Elapsed Minutes Reads Processed Reads/Minute 01:26:48.143 INFO CountReads - 7600360 read(s) filtered by: WellformedReadFilter 01:26:48.146 INFO ProgressMeter - unmapped 0.2 0 0.0 01:26:48.147 INFO ProgressMeter - Traversal complete. Processed 0 total reads in 0.2 minutes. 01:26:48.147 INFO CountReads - CountReads counted 0 total reads 01:26:48.147 INFO CountReads - Shutting down engine [June 24, 2020 1:26:48 AM CDT] org.broadinstitute.hellbender.tools.CountReads done. Elapsed time: 0.18 minutes. Runtime.totalMemory()=2804940800 Tool returned: 0 |
What in all that are we actually looking for you might ask?
01:26:48.143 INFO CountReads - 7600360 read(s) filtered by: WellformedReadFilter 01:26:48.147 INFO ProgressMeter - Traversal complete. Processed 0 total reads in 0.2 minutes. |
This tells us that the bam file contains 7600360 total reads and that none were removed by any filtering options. The lack of anything being removed should make sense since we didn't try to filter anything out.
grep -c "^+$" $SCRATCH/GVA_bowtie2_mapping/SRR030257_1.fastq grep -c "^+$" $SCRATCH/GVA_bowtie2_mapping/SRR030257_2.fastq |
While we haven't discussed sam format in much detail, each read gets its own line, and if you compare the .sam file and the original fastq file listed above, you see that each line on the sam file seems to start with 'SRR030257' followed by a number related to the read. This gives us a base handle to check with grep
grep -c "^SRR030257" SRR030257.sam |
In the case of the sam file, you can see that the 7600360 exactly matches what we saw on the GATK output, while the fastq files each show 3800180 (or 1/2 the total reads). While this is somewhat trivial and
As mentioned this is a very small introduction to GATK adapted from one of the broad's tutorials which can be found here http://gatkforums.broadinstitute.org/gatk/discussion/1209/howto-run-the-gatk-for-the-first-time. Feel free to explore that link and the other tutorial links for taking GATK further.