(EXPERIMENTAL) Processes reads from a MITESeq or other saturation mutagenesis experiment.
Category Coverage Analysis
Process reads from a saturation mutagenesis experiment.
This tool processes reads from an experiment that systematically perturbs a mini-gene to ascertain which amino-acid variations are tolerable at each codon of the open reading frame. It's main job is to discover variations from wild-type sequence among the reads, and to summarize the variations observed.
- A BAM file. Any combination of paired or unpaired reads can be presented. In paired mode (the default mode of operation of the program where overlapping pairs are combined before variant calling), the BAM must be in unsorted or query-name sorted order (so that read pairs are adjacent). The BAM file should be aligned to the amplicon under scrutiny. You may also specify a text file listing several BAMs.
- A fasta-formatted reference file with the wild-type sequence of the gene as a single contig. It's best to include the entire amplicon including any expected 5' and 3' UTRs.
- A description of the location of the open reading frame (ORF) within the reference. For example, --orf 128-1285 would describe an ORF that begins at reference position 128 (1-based coordinates) and ends at reference position 1285 (inclusive). It's possible (though unlikely) to have an ORF with multiple exons: List the exon ranges separated by commas. For example, --orf 128-1284,1384-1433 would describe an ORF with two exons. The total length of the ORF must be divisible by 3, and should begin with a start codon and end with a stop codon.
- An outputPathAndPrefix that specifies where to write the output reports.
- The most important report is a tab-delimited text file named outputPathAndPrefix.variantCounts which describes the observed variations from reference, the number of times each was observed, the effect of the observed variations on the codons, and their translation into amino acids.
- There are a number of additional reports that summarize this information for each codon.
gatk AnalyzeSaturationMutagenesis \ -I input_reads.bam \ -R referenceGene.fasta \ --orf 128-1285 \ -O /path/to/output/and/reportNamePrefix
This Read Filter is automatically applied to the data by the Engine before processing by AnalyzeSaturationMutagenesis.
AnalyzeSaturationMutagenesis specific arguments
This table summarizes the command-line arguments that are specific to this tool. For more details on each argument, see the list further down below the table or click on an argument name to jump directly to that entry in the list.
|Argument name(s)||Default value||Summary|
|BAM/SAM/CRAM file containing reads|
||reference interval(s) of the ORF (1-based, inclusive), for example, '134-180,214-238' (no spaces)|
|output file prefix|
|Reference sequence file|
|Optional Tool Arguments|
||read one or more arguments files and add them to the command line|
|-1||Size of the cloud-only prefetch buffer (in MB; 0 to disable). Defaults to cloudPrefetchBuffer if unset.|
|40||Size of the cloud-only prefetch buffer (in MB; 0 to disable).|
||KNKNTTTTRSRSIIMIQHQHPPPPRRRRLLLLEDEDAAAAGGGGVVVVXYXYSSSSXCWCLFLF||codon translation (a string of 64 amino acid codes|
|false||If true, don't cache bam indexes, this will reduce memory requirements but may harm performance if many intervals are specified. Caching is automatically disabled if there are no intervals specified.|
||false||If specified, do not check the sequence dictionaries from our inputs for compatibility. Use at your own risk!|
||false||examine supplemental alignments to find large deletions|
|20||If the GCS bucket channel errors out, how many times it will attempt to re-initiate the connection|
||Project to bill when accessing "requester pays" buckets. If unset, these buckets cannot be accessed. User must have storage.buckets.get permission on the bucket being accessed.|
|false||display the help message|
|ALL||Interval merging rule for abutting intervals|
|One or more genomic intervals over which to operate|
||15||minimum length of supplemental alignment|
||2||minimum number of wt calls flanking variant|
||15||minimum size of high-quality portion of read|
||4||minimum map quality for read alignment. reads having alignments with MAPQs less than this are treated as unmapped.|
||30||minimum quality score for analyzed portion of read|
||3||minimum number of observations of reported variants|
||true||paired mode evaluation of variants (combine mates, when possible)|
||false||If true, don't emit genotype fields when writing vcf file output.|
||false||display the version number for this tool|
||false||write BAM of rejected reads|
|Optional Common Arguments|
||true||If true, adds a PG tag to created SAM/BAM/CRAM files.|
||true||If true, adds a command line header line to created VCF files.|
|true||If true, create a BAM/CRAM index when writing a coordinate-sorted BAM/CRAM file.|
|false||If true, create a MD5 digest for any BAM/SAM/CRAM file created|
|true||If true, create a VCF index when writing a coordinate-sorted VCF file.|
|false||If true, create a a MD5 digest any VCF file created.|
|Read filters to be disabled before analysis|
||false||Disable all tool default read filters (WARNING: many tools will not function correctly without their default read filters on)|
|One or more genomic intervals to exclude from processing|
||A configuration file to use with the GATK.|
|0||Amount of padding (in bp) to add to each interval you are excluding.|
|0||Amount of padding (in bp) to add to each interval you are including.|
|UNION||Set merging approach to use for combining interval inputs|
|false||Lenient processing of VCF files|
||false||Whether to suppress job-summary info on System.err.|
|Read filters to be applied before analysis|
||Indices to use for the read inputs. If specified, an index must be provided for every read input and in the same order as the read inputs. If this argument is not specified, the path to the index for each input will be inferred automatically.|
|SILENT||Validation stringency for all SAM/BAM/CRAM/SRA files read by this program. The default stringency value SILENT can improve performance when processing a BAM file in which variable-length data (read, qualities, tags) do not otherwise need to be decoded.|
||10.0||Output traversal statistics every time this many seconds elapse|
||Use the given sequence dictionary as the master/canonical sequence dictionary. Must be a .dict file.|
||Temp directory to use.|
|false||Whether to use the JdkDeflater (as opposed to IntelDeflater)|
|false||Whether to use the JdkInflater (as opposed to IntelInflater)|
||INFO||Control verbosity of logging.|
||false||display hidden arguments|
Arguments in this list are specific to this tool. Keep in mind that other arguments are available that are shared with other tools (e.g. command-line GATK arguments); see Inherited arguments above.
--add-output-sam-program-record / -add-output-sam-program-record
If true, adds a PG tag to created SAM/BAM/CRAM files.
--add-output-vcf-command-line / -add-output-vcf-command-line
If true, adds a command line header line to created VCF files.
read one or more arguments files and add them to the command line
--cloud-index-prefetch-buffer / -CIPB
int -1 [ [ -∞ ∞ ] ]
--cloud-prefetch-buffer / -CPB
Size of the cloud-only prefetch buffer (in MB; 0 to disable).
int 40 [ [ -∞ ∞ ] ]
codon translation (a string of 64 amino acid codes
--create-output-bam-index / -OBI
If true, create a BAM/CRAM index when writing a coordinate-sorted BAM/CRAM file.
--create-output-bam-md5 / -OBM
If true, create a MD5 digest for any BAM/SAM/CRAM file created
If true, create a VCF index when writing a coordinate-sorted VCF file.
--create-output-variant-md5 / -OVM
If true, create a a MD5 digest any VCF file created.
--disable-bam-index-caching / -DBIC
--disable-read-filter / -DF
Read filters to be disabled before analysis
--disable-sequence-dictionary-validation / -disable-sequence-dictionary-validation
--disable-tool-default-read-filters / -disable-tool-default-read-filters
--exclude-intervals / -XL
One or more genomic intervals to exclude from processing
Use this argument to exclude certain parts of the genome from the analysis (like -L, but the opposite). This argument can be specified multiple times. You can use samtools-style intervals either explicitly on the command line (e.g. -XL 1 or -XL 1:100-200) or by loading in a file containing a list of intervals (e.g. -XL myFile.intervals).
examine supplemental alignments to find large deletions
A configuration file to use with the GATK.
--gcs-max-retries / -gcs-retries
If the GCS bucket channel errors out, how many times it will attempt to re-initiate the connection
int 20 [ [ -∞ ∞ ] ]
Project to bill when accessing "requester pays" buckets. If unset, these buckets cannot be accessed. User must have storage.buckets.get permission on the bucket being accessed.
--help / -h
display the help message
--input / -I
BAM/SAM/CRAM file containing reads
R List[GATKPath] 
--interval-exclusion-padding / -ixp
Amount of padding (in bp) to add to each interval you are excluding.
Use this to add padding to the intervals specified using -XL. For example, '-XL 1:100' with a padding value of 20 would turn into '-XL 1:80-120'. This is typically used to add padding around targets when analyzing exomes.
int 0 [ [ -∞ ∞ ] ]
--interval-merging-rule / -imr
Interval merging rule for abutting intervals
By default, the program merges abutting intervals (i.e. intervals that are directly side-by-side but do not actually overlap) into a single continuous interval. However you can change this behavior if you want them to be treated as separate intervals instead.
The --interval-merging-rule argument is an enumerated type (IntervalMergingRule), which can have one of the following values:
--interval-padding / -ip
Amount of padding (in bp) to add to each interval you are including.
Use this to add padding to the intervals specified using -L. For example, '-L 1:100' with a padding value of 20 would turn into '-L 1:80-120'. This is typically used to add padding around targets when analyzing exomes.
int 0 [ [ -∞ ∞ ] ]
--interval-set-rule / -isr
Set merging approach to use for combining interval inputs
By default, the program will take the UNION of all intervals specified using -L and/or -XL. However, you can change this setting for -L, for example if you want to take the INTERSECTION of the sets instead. E.g. to perform the analysis only on chromosome 1 exomes, you could specify -L exomes.intervals -L 1 --interval-set-rule INTERSECTION. However, it is not possible to modify the merging approach for intervals passed using -XL (they will always be merged using UNION). Note that if you specify both -L and -XL, the -XL interval set will be subtracted from the -L interval set.
The --interval-set-rule argument is an enumerated type (IntervalSetRule), which can have one of the following values:
- Take the union of all intervals
- Take the intersection of intervals (the subset that overlaps all intervals specified)
--intervals / -L
One or more genomic intervals over which to operate
--lenient / -LE
Lenient processing of VCF files
minimum length of supplemental alignment
int 15 [ [ -∞ ∞ ] ]
minimum number of wt calls flanking variant
int 2 [ [ -∞ ∞ ] ]
minimum size of high-quality portion of read
int 15 [ [ -∞ ∞ ] ]
minimum map quality for read alignment. reads having alignments with MAPQs less than this are treated as unmapped.
int 4 [ [ -∞ ∞ ] ]
minimum quality score for analyzed portion of read
int 30 [ [ -∞ ∞ ] ]
minimum number of observations of reported variants
long 3 [ [ -∞ ∞ ] ]
reference interval(s) of the ORF (1-based, inclusive), for example, '134-180,214-238' (no spaces)
R String null
--output-file-prefix / -O
output file prefix
R String null
paired mode evaluation of variants (combine mates, when possible)
Whether to suppress job-summary info on System.err.
--read-filter / -RF
Read filters to be applied before analysis
--read-index / -read-index
The --read-validation-stringency argument is an enumerated type (ValidationStringency), which can have one of the following values:
--reference / -R
Reference sequence file
R GATKPath null
--seconds-between-progress-updates / -seconds-between-progress-updates
Output traversal statistics every time this many seconds elapse
double 10.0 [ [ -∞ ∞ ] ]
--sequence-dictionary / -sequence-dictionary
Use the given sequence dictionary as the master/canonical sequence dictionary. Must be a .dict file.
--showHidden / -showHidden
display hidden arguments
If true, don't emit genotype fields when writing vcf file output.
Temp directory to use.
--use-jdk-deflater / -jdk-deflater
Whether to use the JdkDeflater (as opposed to IntelDeflater)
--use-jdk-inflater / -jdk-inflater
Whether to use the JdkInflater (as opposed to IntelInflater)
--verbosity / -verbosity
Control verbosity of logging.
The --verbosity argument is an enumerated type (LogLevel), which can have one of the following values:
display the version number for this tool
write BAM of rejected reads
GATK version 126.96.36.199-SNAPSHOT built at Sun, 2 Aug 2020 22:56:07 -0400.