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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:
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| 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.
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Tutorial: Use GATK to count the number of reads in a bam file
Using the above 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 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.
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Don't forget you will still need to start your command with java -jar /opt/apps/gatk/3.5.0/GenomeAnalysisTK.jar to envoke java and gatk before specifying your arguments. |
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java -jar /opt/apps/gatk/3.5.0/GenomeAnalysisTK.jar -T CountLoci -R exampleFASTA.fasta -I exampleBAM.bam |
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gatk CountReads -R NC_012967.1.fasta -I SRR030257.bam |
Did you get the result you expected?
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The foloowing line tells us things did not go as intended. A USER ERROR has occurred: Fasta dict file file:///$SCRATCH/GVA_GATK/NC_012967.1.dict for reference file:///$SCRATCH/GVA_GATK/NC_012967.1.fasta does not exist. Please see http://gatkforums.broadinstitute.org/discussion/1601/how-can-i-prepare-a-fasta-file-to-use-as-reference for help creating it |
In following that very helpful link you'll see 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.
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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):
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Using GATK jar /opt/tacc_mounts/home1/01821/ded/local/bin/gatk-package-4.1.2.0-local.jar
Running:
java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /opt/tacc_mounts/home1/01821/ded/local/bin/gatk-package-4.1.2.0-local.jar CountReads -R NC_012967.1.fasta -I SRR030257.bam
09:27:58.776 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/opt/tacc_mounts/home1/01821/ded/local/bin/gatk-package-4.1.2.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
May 31, 2019 9:27:59 AM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
09:27:59.973 INFO CountReads - ------------------------------------------------------------
09:27:59.973 INFO CountReads - The Genome Analysis Toolkit (GATK) v4.1.2.0
09:27:59.973 INFO CountReads - For support and documentation go to https://software.broadinstitute.org/gatk/
09:27:59.973 INFO CountReads - Executing as ded@login1 on Linux v4.4.103-6.38-default amd64
09:27:59.973 INFO CountReads - Java runtime: OpenJDK 64-Bit Server VM v1.8.0_151-b12
09:27:59.973 INFO CountReads - Start Date/Time: May 31, 2019 9:27:58 AM CDT
09:27:59.973 INFO CountReads - ------------------------------------------------------------
09:27:59.973 INFO CountReads - ------------------------------------------------------------
09:27:59.974 INFO CountReads - HTSJDK Version: 2.19.0
09:27:59.974 INFO CountReads - Picard Version: 2.19.0
09:27:59.974 INFO CountReads - HTSJDK Defaults.COMPRESSION_LEVEL : 2
09:27:59.974 INFO CountReads - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
09:27:59.974 INFO CountReads - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
09:27:59.974 INFO CountReads - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
09:27:59.974 INFO CountReads - Deflater: IntelDeflater
09:27:59.974 INFO CountReads - Inflater: IntelInflater
09:27:59.974 INFO CountReads - GCS max retries/reopens: 20
09:27:59.974 INFO CountReads - Requester pays: disabled
09:27:59.974 INFO CountReads - Initializing engine
09:28:00.322 INFO CountReads - Done initializing engine
09:28:00.322 INFO ProgressMeter - Starting traversal
09:28:00.322 INFO ProgressMeter - Current Locus Elapsed Minutes Reads Processed Reads/Minute
09:28:11.059 INFO CountReads - 7600360 read(s) filtered by: WellformedReadFilter
09:28:11.061 INFO ProgressMeter - unmapped 0.2 0 0.0
09:28:11.062 INFO ProgressMeter - Traversal complete. Processed 0 total reads in 0.2 minutes.
09:28:11.062 INFO CountReads - Shutting down engine
[May 31, 2019 9:28:11 AM CDT] org.broadinstitute.hellbender.tools.CountReads done. Elapsed time: 0.21 minutes.
Runtime.totalMemory()=2847932416
Tool returned:
0 |
What in all that are we actually looking for you might ask?
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09:28:11.059 INFO 13:05:21,093 MicroScheduler - 0 reads were filtered out during the traversal out of approximately 33CountReads - 7600360 read(s) filtered by: WellformedReadFilter 09:28:11.062 INFO ProgressMeter - Traversal complete. Processed 0 total reads (in 0.00%)2 minutes. |
This tells us that the bam file contains 33 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. Can you figure out why this might be different than the total 9555594 reads that were present in the original fastq files SRR030257_1.fastq and SRR030257_1.fastq (check them in your GVA_bowtie2_mapping directory)?
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The .bam file (and .sam file only deal with reads that correctly mapped back to to the genome within the bounds of the bowtie2 mapping program. This means the 'missing reads did not map. Perhaps they are adapter dimers or reads that have adapters present on the ends causing mapping problems (could be checked with fastQC fairly simply), Perhaps they are a plasmid not present in the reference genome (could try to assemble this plasmid with SPAdes). |
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.
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