PairReadsQSeq

PairReadsQSeq (PRQ) is a Hadoop utility to convert Illumina Qseq or Fastq files into Prq file format; prq files are simply 5 tab-separated fields per line: id, read 1, base qualities 1, read 2, base qualities 2.

PairReadsQSeq standardizes base quality scores to Sanger-style Phred+33 encoding. In addition, it converts unknown bases to ‘N’ (as opposed to the ‘.’ used in QSeq files). In any case, PRQ’s main purpose is to place both read and mate in the same data record.

PairReadsQSeq by default filters read pairs where both reads don’t have a minimum number of known bases (30 by default).

In addition, PairReadsQSeq by default filters read pairs if both its reads failed the machine quality checks (that would be the last column of the Qseq file format, or the Y/N flag in Illumina Fastq records).

Usage

To run PairReadsQSeq, launch seal prq. For example,

seal prq /user/me/qseq_input /user/me/prq_output

seal_prq follows the normal Seal usage convention. See the section Program Usage for details.

Input format

By default PairReadsQSeq expects input in Qseq format. You can specify Fastq by setting –input-format fastq:

seal prq --input-format fastq fastq_input prq_output

Quality encoding

PairReadsQSeq expects the standard base quality encoding scheme for each format: Illumina Phred+64 for qseq and Sanger Phred+33 for fastq. If you need to override this default behaviour set the seal.input.base-quality-encoding property. With qseq data use:

seal prq --input-format qseq -D seal.input.base-quality-encoding=sanger qseq_input prq_output

With fastq data use:

seal prq --input-format fastq  -D seal.input.base-quality-encoding=illumina fastq_input prq_output

The quality encoding will always be encoded in Sanger Phred+33 format in the output prq file.

Configurable Properties

Name	Meaning
seal.prq.input-format	“qseq” or “fastq”; equivalent to the --input-format argument.
seal.input.base-quality-encoding	“illumina” or “sanger”
seal.prq.min-bases-per-read	See Read Filtering
seal.prq.drop-failed-filter	See Read Filtering
seal.prq.warning-only-if-unpaired	PRQ normally stops with an error if it finds an unpaired read. If this property is set to true it will instead emit a warning and keep going.

In addition, all the general Seal and Hadoop configuration properties apply.

Note

Config File Section Title: Prq

Deprecated Properties

The following properties, recognized in previous versions of Seal, have been deprecated and replaced. They are still functional for the moment, but will be completely removed in future versions so you are urged to update your configurations and scripts.

Deprecated property	Replacement
bl.prq.min-bases-per-read	seal.prq.min-bases-per-read
bl.prq.drop-failed-filter	seal.prq.drop-failed-filter
bl.prq.warning-only-if-unpaired	seal.prq.warning-only-if-unpaired

Read Filtering

PairReadsQSeq can filter read pairs that fail to meet certain quality criteria.

• not enough known bases;
• failure to meet the sequencing machine’s quality checks.

Min number of known bases

Property name: seal.prq.min-bases-per-read

Reads output from the sequencing machine often contain bases that could not be read. Reads with too few known bases are undesirable, so PairReadsQSeq can filter them. By default, if neither read in a pair has at least 30 known bases the pair is dropped. You can override this setting by setting the seal.prq.min-bases-per-read property to your desired value. For instance, to require 15 known bases:

seal prq -D seal.prq.min-bases-per-read=15 /user/me/qseq_data /user/me/prq_data

To disable this feature specify a minimum known base threshold of 0.

Failed quality checks

Property name: seal.prq.drop-failed-filter

As previously mentioned, PairReadsQSeq by default filters read pairs if both the pair’s reads failed the machine quality checks. Reads that don’t meet machine-based quality checks are identified in qseq files by the value in the last column (0: failed check; 1: passed check), and in fastq files the Y/N filtered flag. To disable filtering behaviour in PairReadsQSeq set the property seal.prq.drop-failed-filter to false.

Counters

PRQ provides a number of counters that report on the number of reads filtered.

NotEnoughBases:number of reads that have fewer known bases than the minimum requirement. FailedFilter:number of reads that failed machine quality checks. Unpaired:number of unpaired reads found in the data (only if seal.prq.warning-only-if-unpaired is enabled). Dropped:number of reads dropped from the dataset for any of the reasons above.