pipelines-docs

Read Groups

A read group (@RG) is a unique identifier that group reads together, capturing relevant information about the sample and the sequencing process and technology, utilized by various downstream bioinformatics tools.

The relevant fields in defining a read group include:

• ID (Identifier): A unique identifier for the read group within the BAM file and across multiple BAM files used in the same dataset.
• SM (Sample): The sample to which the reads belong.
• PL (Platform): The technology used to sequence the reads (e.g., PACBIO).
• PM (Platform Model): The platform model reflecting the instrument series (e.g., REVIO, ASTRO, SEQUEL, RS).
• PU (Platform Unit): A unique identifier for the sequencer unit used for sequencing (i.e., PacBio movie name).
• LB (Library): The library used to sequence the reads.
• DS (Description): Semantic information about the reads in the group, encoded as a semicolon-delimited list of “Key=Value” strings.

Mandatory Description (DS) Information

Key	Value Specification	Example
READTYPE	One of ZMW, HQREGION, SUBREAD, CCS, SCRAP, or UNKNOWN	CCS
BINDINGKIT	Binding kit part number	102-739-100
SEQUENCINGKIT	Sequencing kit part number	102-118-800
BASECALLERVERSION	Basecaller version number	5.0
FRAMERATEHZ	Frame rate in Hz	100
CONTROL	TRUE if reads are classified as spike-in controls, otherwise CONTROL key is absent	TRUE

Optional Description (DS) Information

Key	Value Specification	Example
BarcodeFile	Name of the FASTA file containing the sequences of the barcodes used	m84046_230828_225743_s2.barcodes.fasta
BarcodeHash	The MD5 hash of the contents of the barcoding sequence file	e7c4279103df8c8de7036efdbdca9008
BarcodeCount	The number of barcode sequences in the barcode file	113
BarcodeMode	Experimental design of the barcodes. Must be Symmetric/Asymmetric/Tailed or None Symmetric	Symmetric
BarcodeQuality	The type of value encoded by the bq tag. Must be Score/Probability/None	Score

Assigning Read Groups

During the alignment process using pbmm2, the original read groups from the unaligned BAM files are linked and maintained in the corresponding alignment BAM files. In-house bash code that utilizes samtools replaces SM and LB information with the correct identifiers used by the portal, as follows:

• SM: <sample name>
• LB: <sample name>.<library>

E.g., in BAM file:

@RG	ID:f115ea06/25--25	PL:PACBIO	DS:READTYPE=CCS;Ipd:Frames=ip;PulseWidth:Frames=pw;BINDINGKIT=102-739-100;SEQUENCINGKIT=102-118-800;BASECALLERVERSION=5.0;FRAMERATEHZ=100.000000;BarcodeFile=metadata/m84046_230828_225743_s2.barcodes.fasta;BarcodeHash=e7c4279103df8c8de7036efdbdca9008;BarcodeCount=113;BarcodeMode=Symmetric;BarcodeQuality=Score	LB:SMACUBS146SV.SMALIRA4HLNS	PU:m84046_230828_225743_s2	SM:SMACUBS146SV	PM:REVIO	BC:ACGCACGTACGAGTAT	CM:R/P1-C1/5.0-25M

Source Code

All the relevant code is accessible in the GitHub repository:

• ReplaceReadGroups.sh [ReplaceReadGroups]

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