UmiAwareMarkDuplicatesWithMateCigar (Picard) (EXPERIMENTAL) – GATK

Identifies duplicate reads using information from read positions and UMIs.

This tool locates and tags duplicate reads in a BAM or SAM file, where duplicate reads aredefined as originating from a single fragment of DNA. It is based on the {@link MarkDuplicatesWithMateCigar} tool, with added logicto leverage Unique Molecular Identifier (UMI) information.

It makes use of the fact that duplicate sets with UMIs can be broken up into subsets based oninformation contained in the UMI. In addition to assuming that all members of a duplicate set must have the same start and end position, it imposes thatthey must also have sufficiently similar UMIs. In this context, 'sufficiently similar' is parameterized by the command lineargument MAX_EDIT_DISTANCE_TO_JOIN, which sets the edit distance between UMIs that will be considered to be part of the sameoriginal molecule. This logic allows for sequencing errors in UMIs.

If UMIs contain dashes, the dashes will be ignored. If UMIs contain Ns, these UMIs will not contribute to UMI metricsassociated with each record. If the MAX_EDIT_DISTANCE_TO_JOIN allows, UMIs with Ns will be included in the duplicate set andthe UMI metrics associated with each duplicate set. Ns are counted as an edit distance from other bases {ATCG}, but are notconsidered different from each other.

This tool is NOT intended to be used on data without UMIs; for marking duplicates in non-UMI data, see {@link MarkDuplicates} or{@link MarkDuplicatesWithMateCigar}. Mixed data (where some reads have UMIs and others do not) is not supported.

Note also that this tool will not work with alignments that have large gaps or deletions, such as those from RNA-seq data.This is due to the need to buffer small genomic windows to ensure integrity of the duplicate marking, while large skips(ex. skipping introns) in the alignment records would force making that window very large, thus exhausting memory.

Note: Metrics labeled as percentages are actually expressed as fractions!

Usage example:

java -jar picard.jar UmiAwareMarkDuplicatesWithMateCigar 
      I=input.bam 
      O=output.bam 
      M=output_duplicate_metrics.txt 
      UMI_METRICS=output_umi_metrics.txt

Category Read Data Manipulation

Overview

This tool locates and tags duplicate reads in a BAM or SAM file, where duplicate reads are defined as originating from a single fragment of DNA. It is based on the MarkDuplicatesWithMateCigar tool, with added logic to leverage Unique Molecular Identifier (UMI) information.

It makes use of the fact that duplicate sets with UMIs can be broken up into subsets based on information contained in the UMI. In addition to assuming that all members of a duplicate set must have the same start and end position, it imposes that they must also have sufficiently similar UMIs. In this context, 'sufficiently similar' is parameterized by the command line argument MAX_EDIT_DISTANCE_TO_JOIN, which sets the edit distance between UMIs that will be considered to be part of the same original molecule. This logic allows for sequencing errors in UMIs.

If UMIs contain dashes, the dashes will be ignored. If UMIs contain Ns, these UMIs will not contribute to UMI metrics associated with each record. If the MAX_EDIT_DISTANCE_TO_JOIN allows, UMIs with Ns will be included in the duplicate set and the UMI metrics associated with each duplicate set. Ns are counted as an edit distance from other bases {ATCG}, but are not considered different from each other.

This tool is NOT intended to be used on data without UMIs; for marking duplicates in non-UMI data, see MarkDuplicates or MarkDuplicatesWithMateCigar. Mixed data (where some reads have UMIs and others do not) is not supported.

Note also that this tool will not work with alignments that have large gaps or deletions, such as those from RNA-seq data. This is due to the need to buffer small genomic windows to ensure integrity of the duplicate marking, while large skips (ex. skipping introns) in the alignment records would force making that window very large, thus exhausting memory.

Note: Metrics labeled as percentages are actually expressed as fractions!

Usage example:


 java -jar picard.jar UmiAwareMarkDuplicatesWithMateCigar \\

      I=input.bam \\

      O=output.bam \\

      M=output_duplicate_metrics.txt \\

      UMI_METRICS=output_umi_metrics.txt

UmiAwareMarkDuplicatesWithMateCigar (Picard) 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
Required Arguments
--INPUT -I		One or more input SAM or BAM files to analyze. Must be coordinate sorted.
--METRICS_FILE -M		File to write duplication metrics to
--OUTPUT -O		The output file to write marked records to
--UMI_METRICS_FILE -UMI_METRICS		UMI Metrics
Optional Tool Arguments
--ALLOW_MISSING_UMIS	false	FOR TESTING ONLY: allow for missing UMIs if data doesn't have UMIs. This option is intended to be used ONLY for testing the code. Use MarkDuplicatesWithMateCigar if data has no UMIs. Mixed data (where some reads have UMIs and others do not) is not supported.
--arguments_file		read one or more arguments files and add them to the command line
--ASSUME_SORT_ORDER -ASO		If not null, assume that the input file has this order even if the header says otherwise.
--BARCODE_TAG		Barcode SAM tag (ex. BC for 10X Genomics)
--CLEAR_DT	true	Clear DT tag from input SAM records. Should be set to false if input SAM doesn't have this tag. Default true
--COMMENT -CO		Comment(s) to include in the output file's header.
--DUPLEX_UMI	false	Treat UMIs as being duplex stranded. This option requires that the UMI consist of two equal length strings that are separated by a hyphen (e.g. 'ATC-GTC'). Reads are considered duplicates if, in addition to standard definition, have identical normalized UMIs. A UMI from the 'bottom' strand is normalized by swapping its content around the hyphen (eg. ATC-GTC becomes GTC-ATC). A UMI from the 'top' strand is already normalized as it is. Both reads from a read pair considered top strand if the read 1 unclipped 5' coordinate is less than the read 2 unclipped 5' coordinate. All chimeric reads and read fragments are treated as having come from the top strand. With this option is it required that the BARCODE_TAG hold non-normalized UMIs. Default false.
--DUPLICATE_SCORING_STRATEGY -DS	SUM_OF_BASE_QUALITIES	The scoring strategy for choosing the non-duplicate among candidates.
--help -h	false	display the help message
--MAX_EDIT_DISTANCE_TO_JOIN	1	Largest edit distance that UMIs must have in order to be considered as coming from distinct source molecules.
--MAX_FILE_HANDLES_FOR_READ_ENDS_MAP -MAX_FILE_HANDLES	8000	Maximum number of file handles to keep open when spilling read ends to disk. Set this number a little lower than the per-process maximum number of file that may be open. This number can be found by executing the 'ulimit -n' command on a Unix system.
--MAX_OPTICAL_DUPLICATE_SET_SIZE	300000	This number is the maximum size of a set of duplicate reads for which we will attempt to determine which are optical duplicates. Please be aware that if you raise this value too high and do encounter a very large set of duplicate reads, it will severely affect the runtime of this tool. To completely disable this check, set the value to -1.
--MAX_SEQUENCES_FOR_DISK_READ_ENDS_MAP -MAX_SEQS	50000	This option is obsolete. ReadEnds will always be spilled to disk.
--MOLECULAR_IDENTIFIER_TAG		SAM tag to uniquely identify the molecule from which a read was derived. Use of this option requires that the BARCODE_TAG option be set to a non null value. Default null.
--OPTICAL_DUPLICATE_PIXEL_DISTANCE	100	The maximum offset between two duplicate clusters in order to consider them optical duplicates. The default is appropriate for unpatterned versions of the Illumina platform. For the patterned flowcell models, 2500 is moreappropriate. For other platforms and models, users should experiment to find what works best.
--PROGRAM_GROUP_COMMAND_LINE -PG_COMMAND		Value of CL tag of PG record to be created. If not supplied the command line will be detected automatically.
--PROGRAM_GROUP_NAME -PG_NAME	UmiAwareMarkDuplicatesWithMateCigar	Value of PN tag of PG record to be created.
--PROGRAM_GROUP_VERSION -PG_VERSION		Value of VN tag of PG record to be created. If not specified, the version will be detected automatically.
--PROGRAM_RECORD_ID -PG	MarkDuplicates	The program record ID for the @PG record(s) created by this program. Set to null to disable PG record creation. This string may have a suffix appended to avoid collision with other program record IDs.
--READ_NAME_REGEX		MarkDuplicates can use the tile and cluster positions to estimate the rate of optical duplication in addition to the dominant source of duplication, PCR, to provide a more accurate estimation of library size. By default (with no READ_NAME_REGEX specified), MarkDuplicates will attempt to extract coordinates using a split on ':' (see Note below). Set READ_NAME_REGEX to 'null' to disable optical duplicate detection. Note that without optical duplicate counts, library size estimation will be less accurate. If the read name does not follow a standard Illumina colon-separation convention, but does contain tile and x,y coordinates, a regular expression can be specified to extract three variables: tile/region, x coordinate and y coordinate from a read name. The regular expression must contain three capture groups for the three variables, in order. It must match the entire read name. e.g. if field names were separated by semi-colon (';') this example regex could be specified (?:.;)?([0-9]+)[^;];([0-9]+)[^;];([0-9]+)[^;]$ Note that if no READ_NAME_REGEX is specified, the read name is split on ':'. For 5 element names, the 3rd, 4th and 5th elements are assumed to be tile, x and y values. For 7 element names (CASAVA 1.8), the 5th, 6th, and 7th elements are assumed to be tile, x and y values.
--READ_ONE_BARCODE_TAG		Read one barcode SAM tag (ex. BX for 10X Genomics)
--READ_TWO_BARCODE_TAG		Read two barcode SAM tag (ex. BX for 10X Genomics)
--REMOVE_DUPLICATES	false	If true do not write duplicates to the output file instead of writing them with appropriate flags set.
--REMOVE_SEQUENCING_DUPLICATES	false	If true remove 'optical' duplicates and other duplicates that appear to have arisen from the sequencing process instead of the library preparation process, even if REMOVE_DUPLICATES is false. If REMOVE_DUPLICATES is true, all duplicates are removed and this option is ignored.
--SORTING_COLLECTION_SIZE_RATIO	0.25	This number, plus the maximum RAM available to the JVM, determine the memory footprint used by some of the sorting collections. If you are running out of memory, try reducing this number.
--TAG_DUPLICATE_SET_MEMBERS	false	If a read appears in a duplicate set, add two tags. The first tag, DUPLICATE_SET_SIZE_TAG (DS), indicates the size of the duplicate set. The smallest possible DS value is 2 which occurs when two reads map to the same portion of the reference only one of which is marked as duplicate. The second tag, DUPLICATE_SET_INDEX_TAG (DI), represents a unique identifier for the duplicate set to which the record belongs. This identifier is the index-in-file of the representative read that was selected out of the duplicate set.
--TAGGING_POLICY	DontTag	Determines how duplicate types are recorded in the DT optional attribute.
--UMI_TAG_NAME	RX	Tag name to use for UMI
--version	false	display the version number for this tool
Optional Common Arguments
--ADD_PG_TAG_TO_READS	true	Add PG tag to each read in a SAM or BAM
--COMPRESSION_LEVEL	5	Compression level for all compressed files created (e.g. BAM and VCF).
--CREATE_INDEX	false	Whether to create an index when writing VCF or coordinate sorted BAM output.
--CREATE_MD5_FILE	false	Whether to create an MD5 digest for any BAM or FASTQ files created.
--GA4GH_CLIENT_SECRETS	client_secrets.json	Google Genomics API client_secrets.json file path.
--MAX_RECORDS_IN_RAM	500000	When writing files that need to be sorted, this will specify the number of records stored in RAM before spilling to disk. Increasing this number reduces the number of file handles needed to sort the file, and increases the amount of RAM needed.
--QUIET	false	Whether to suppress job-summary info on System.err.
--REFERENCE_SEQUENCE -R		Reference sequence file.
--TMP_DIR		One or more directories with space available to be used by this program for temporary storage of working files
--USE_JDK_DEFLATER -use_jdk_deflater	false	Use the JDK Deflater instead of the Intel Deflater for writing compressed output
--USE_JDK_INFLATER -use_jdk_inflater	false	Use the JDK Inflater instead of the Intel Inflater for reading compressed input
--VALIDATION_STRINGENCY	STRICT	Validation stringency for all SAM files read by this program. Setting stringency to SILENT can improve performance when processing a BAM file in which variable-length data (read, qualities, tags) do not otherwise need to be decoded.
--VERBOSITY	INFO	Control verbosity of logging.
Advanced Arguments
--showHidden	false	display hidden arguments
Deprecated Arguments
--ASSUME_SORTED -AS	false	If true, assume that the input file is coordinate sorted even if the header says otherwise. Deprecated, used ASSUME_SORT_ORDER=coordinate instead.

Argument details

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_PG_TAG_TO_READS

Add PG tag to each read in a SAM or BAM

boolean true

--ALLOW_MISSING_UMIS

FOR TESTING ONLY: allow for missing UMIs if data doesn't have UMIs. This option is intended to be used ONLY for testing the code. Use MarkDuplicatesWithMateCigar if data has no UMIs. Mixed data (where some reads have UMIs and others do not) is not supported.

boolean false

--arguments_file

read one or more arguments files and add them to the command line

List[File] []

--ASSUME_SORT_ORDER / -ASO

If not null, assume that the input file has this order even if the header says otherwise.

Exclusion: This argument cannot be used at the same time as ASSUME_SORTED.

The --ASSUME_SORT_ORDER argument is an enumerated type (SortOrder), which can have one of the following values:

unsorted
queryname
coordinate
duplicate
unknown

SortOrder null

--ASSUME_SORTED / -AS

If true, assume that the input file is coordinate sorted even if the header says otherwise. Deprecated, used ASSUME_SORT_ORDER=coordinate instead.

Exclusion: This argument cannot be used at the same time as ASSUME_SORT_ORDER.

boolean false

--BARCODE_TAG

Barcode SAM tag (ex. BC for 10X Genomics)

String null

--CLEAR_DT

Clear DT tag from input SAM records. Should be set to false if input SAM doesn't have this tag. Default true

boolean true

--COMMENT / -CO

Comment(s) to include in the output file's header.

List[String] []

--COMPRESSION_LEVEL

Compression level for all compressed files created (e.g. BAM and VCF).

int 5 [ [ -∞ ∞ ] ]

--CREATE_INDEX

Whether to create an index when writing VCF or coordinate sorted BAM output.

Boolean false

--CREATE_MD5_FILE

Whether to create an MD5 digest for any BAM or FASTQ files created.

boolean false

--DUPLEX_UMI

Treat UMIs as being duplex stranded. This option requires that the UMI consist of two equal length strings that are separated by a hyphen (e.g. 'ATC-GTC'). Reads are considered duplicates if, in addition to standard definition, have identical normalized UMIs. A UMI from the 'bottom' strand is normalized by swapping its content around the hyphen (eg. ATC-GTC becomes GTC-ATC). A UMI from the 'top' strand is already normalized as it is. Both reads from a read pair considered top strand if the read 1 unclipped 5' coordinate is less than the read 2 unclipped 5' coordinate. All chimeric reads and read fragments are treated as having come from the top strand. With this option is it required that the BARCODE_TAG hold non-normalized UMIs. Default false.

boolean false

--DUPLICATE_SCORING_STRATEGY / -DS

The scoring strategy for choosing the non-duplicate among candidates.

The --DUPLICATE_SCORING_STRATEGY argument is an enumerated type (ScoringStrategy), which can have one of the following values:

SUM_OF_BASE_QUALITIES
TOTAL_MAPPED_REFERENCE_LENGTH
RANDOM

ScoringStrategy SUM_OF_BASE_QUALITIES

--GA4GH_CLIENT_SECRETS

Google Genomics API client_secrets.json file path.

String client_secrets.json

--help / -h

display the help message

boolean false

--INPUT / -I

One or more input SAM or BAM files to analyze. Must be coordinate sorted.

R List[String] []

--MAX_EDIT_DISTANCE_TO_JOIN / -MAX_EDIT_DISTANCE_TO_JOIN

Largest edit distance that UMIs must have in order to be considered as coming from distinct source molecules.

int 1 [ [ -∞ ∞ ] ]

--MAX_FILE_HANDLES_FOR_READ_ENDS_MAP / -MAX_FILE_HANDLES

Maximum number of file handles to keep open when spilling read ends to disk. Set this number a little lower than the per-process maximum number of file that may be open. This number can be found by executing the 'ulimit -n' command on a Unix system.

int 8000 [ [ -∞ ∞ ] ]

--MAX_OPTICAL_DUPLICATE_SET_SIZE

This number is the maximum size of a set of duplicate reads for which we will attempt to determine which are optical duplicates. Please be aware that if you raise this value too high and do encounter a very large set of duplicate reads, it will severely affect the runtime of this tool. To completely disable this check, set the value to -1.

long 300000 [ [ -∞ ∞ ] ]

--MAX_RECORDS_IN_RAM

When writing files that need to be sorted, this will specify the number of records stored in RAM before spilling to disk. Increasing this number reduces the number of file handles needed to sort the file, and increases the amount of RAM needed.

Integer 500000 [ [ -∞ ∞ ] ]

--MAX_SEQUENCES_FOR_DISK_READ_ENDS_MAP / -MAX_SEQS

This option is obsolete. ReadEnds will always be spilled to disk.
If more than this many sequences in SAM file, don't spill to disk because there will not be enough file handles.

int 50000 [ [ -∞ ∞ ] ]

--METRICS_FILE / -M

File to write duplication metrics to

R File null

--MOLECULAR_IDENTIFIER_TAG

SAM tag to uniquely identify the molecule from which a read was derived. Use of this option requires that the BARCODE_TAG option be set to a non null value. Default null.

String null

--OPTICAL_DUPLICATE_PIXEL_DISTANCE

The maximum offset between two duplicate clusters in order to consider them optical duplicates. The default is appropriate for unpatterned versions of the Illumina platform. For the patterned flowcell models, 2500 is moreappropriate. For other platforms and models, users should experiment to find what works best.

int 100 [ [ -∞ ∞ ] ]

--OUTPUT / -O

The output file to write marked records to

R File null

--PROGRAM_GROUP_COMMAND_LINE / -PG_COMMAND

Value of CL tag of PG record to be created. If not supplied the command line will be detected automatically.

String null

--PROGRAM_GROUP_NAME / -PG_NAME

Value of PN tag of PG record to be created.

String UmiAwareMarkDuplicatesWithMateCigar

--PROGRAM_GROUP_VERSION / -PG_VERSION

Value of VN tag of PG record to be created. If not specified, the version will be detected automatically.

String null

--PROGRAM_RECORD_ID / -PG

The program record ID for the @PG record(s) created by this program. Set to null to disable PG record creation. This string may have a suffix appended to avoid collision with other program record IDs.

String MarkDuplicates

--QUIET

Whether to suppress job-summary info on System.err.

Boolean false

--READ_NAME_REGEX

MarkDuplicates can use the tile and cluster positions to estimate the rate of optical duplication in addition to the dominant source of duplication, PCR, to provide a more accurate estimation of library size. By default (with no READ_NAME_REGEX specified), MarkDuplicates will attempt to extract coordinates using a split on ':' (see Note below). Set READ_NAME_REGEX to 'null' to disable optical duplicate detection. Note that without optical duplicate counts, library size estimation will be less accurate. If the read name does not follow a standard Illumina colon-separation convention, but does contain tile and x,y coordinates, a regular expression can be specified to extract three variables: tile/region, x coordinate and y coordinate from a read name. The regular expression must contain three capture groups for the three variables, in order. It must match the entire read name. e.g. if field names were separated by semi-colon (';') this example regex could be specified (?:.*;)?([0-9]+)[^;]*;([0-9]+)[^;]*;([0-9]+)[^;]*$ Note that if no READ_NAME_REGEX is specified, the read name is split on ':'. For 5 element names, the 3rd, 4th and 5th elements are assumed to be tile, x and y values. For 7 element names (CASAVA 1.8), the 5th, 6th, and 7th elements are assumed to be tile, x and y values.

String

--READ_ONE_BARCODE_TAG

Read one barcode SAM tag (ex. BX for 10X Genomics)

String null

--READ_TWO_BARCODE_TAG

Read two barcode SAM tag (ex. BX for 10X Genomics)

String null

--REFERENCE_SEQUENCE / -R

Reference sequence file.

File null

--REMOVE_DUPLICATES

If true do not write duplicates to the output file instead of writing them with appropriate flags set.

boolean false

--REMOVE_SEQUENCING_DUPLICATES

If true remove 'optical' duplicates and other duplicates that appear to have arisen from the sequencing process instead of the library preparation process, even if REMOVE_DUPLICATES is false. If REMOVE_DUPLICATES is true, all duplicates are removed and this option is ignored.

boolean false

--showHidden / -showHidden

display hidden arguments

boolean false

--SORTING_COLLECTION_SIZE_RATIO

This number, plus the maximum RAM available to the JVM, determine the memory footprint used by some of the sorting collections. If you are running out of memory, try reducing this number.

double 0.25 [ [ -∞ ∞ ] ]

--TAG_DUPLICATE_SET_MEMBERS

If a read appears in a duplicate set, add two tags. The first tag, DUPLICATE_SET_SIZE_TAG (DS), indicates the size of the duplicate set. The smallest possible DS value is 2 which occurs when two reads map to the same portion of the reference only one of which is marked as duplicate. The second tag, DUPLICATE_SET_INDEX_TAG (DI), represents a unique identifier for the duplicate set to which the record belongs. This identifier is the index-in-file of the representative read that was selected out of the duplicate set.

boolean false

--TAGGING_POLICY

Determines how duplicate types are recorded in the DT optional attribute.

The --TAGGING_POLICY argument is an enumerated type (DuplicateTaggingPolicy), which can have one of the following values:

DontTag
OpticalOnly
All

DuplicateTaggingPolicy DontTag

--TMP_DIR

One or more directories with space available to be used by this program for temporary storage of working files

List[File] []

--UMI_METRICS_FILE / -UMI_METRICS

UMI Metrics

R File null

--UMI_TAG_NAME / -UMI_TAG_NAME

Tag name to use for UMI

String RX

--USE_JDK_DEFLATER / -use_jdk_deflater

Use the JDK Deflater instead of the Intel Deflater for writing compressed output

Boolean false

--USE_JDK_INFLATER / -use_jdk_inflater

Use the JDK Inflater instead of the Intel Inflater for reading compressed input

Boolean false

--VALIDATION_STRINGENCY

Validation stringency for all SAM files read by this program. Setting stringency to SILENT can improve performance when processing a BAM file in which variable-length data (read, qualities, tags) do not otherwise need to be decoded.

The --VALIDATION_STRINGENCY argument is an enumerated type (ValidationStringency), which can have one of the following values:

STRICT
LENIENT
SILENT

ValidationStringency STRICT

--VERBOSITY

Control verbosity of logging.

The --VERBOSITY argument is an enumerated type (LogLevel), which can have one of the following values:

ERROR
WARNING
INFO
DEBUG

LogLevel INFO

--version

display the version number for this tool

boolean false

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GATK version 4.2.2.0-SNAPSHOT built at Thu, 19 Aug 2021 09:49:28 -0700.

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Usage example:

Category Read Data Manipulation

Overview

Usage example:

UmiAwareMarkDuplicatesWithMateCigar (Picard) specific arguments

Argument details

--ASSUME_SORT_ORDER / -ASO

--ASSUME_SORTED / -AS

--COMMENT / -CO

--DUPLICATE_SCORING_STRATEGY / -DS

--help / -h

--INPUT / -I

--MAX_EDIT_DISTANCE_TO_JOIN / -MAX_EDIT_DISTANCE_TO_JOIN

--MAX_FILE_HANDLES_FOR_READ_ENDS_MAP / -MAX_FILE_HANDLES

--MAX_SEQUENCES_FOR_DISK_READ_ENDS_MAP / -MAX_SEQS

--METRICS_FILE / -M

--OUTPUT / -O

--PROGRAM_GROUP_COMMAND_LINE / -PG_COMMAND

--PROGRAM_GROUP_NAME / -PG_NAME

--PROGRAM_GROUP_VERSION / -PG_VERSION

--PROGRAM_RECORD_ID / -PG

--REFERENCE_SEQUENCE / -R

--showHidden / -showHidden

--UMI_METRICS_FILE / -UMI_METRICS

--UMI_TAG_NAME / -UMI_TAG_NAME

--USE_JDK_DEFLATER / -use_jdk_deflater

--USE_JDK_INFLATER / -use_jdk_inflater