UMI Scripts - Applications

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Applications

The following applications are included with this package free of charge.

Fastq applications

These pre-alignment scripts are intended to work on Fastq files and extract a UMI and either append it to the read name or write it as a third file. This is usually done prior to trimming adapters. GZip compressed files are natively handled. Processed reads in some cases can be piped out to downstream applications (adapter trimming or alignment).

• embedded_UMI_extractor.pl

  A simple script for extracting the UMI barcode from the beginning of the read. A fixed sequence may or may not be present between the UMI and the sequenced insert. Both single-end and paired-end (with or without a second UMI in the second read) fastq files are supported. The UMI may added as SAM tags to the Fastq read header, appended to the read name, or written out as an unaligned Sam or Bam file.

• merge_umi_fastq.pl

  A script for merging a third fastq file of UMI sequences with one (single-end) or two (paired-end) fastq reads. The UMI may be appended to the read name (non-standard), added as SAM tags to the Fastq read header, or written out as an unaligned Sam or Bam file, depending on need and available support by the aligner.

• smallRNA_pe_umi_extractor.pl

  A simple script for extracting the adapter and UMI sequence from a paired-end implementation of the Qiagen small RNA library preparation. The UMI and Qiagen adapter is identified in R2, any complete or partial Qiagen adapter identified and removed from R1, and R1 is written to output with the UMI appended to the read name for de-duplication later.

• qiagen_smallRNA_umi_extractor.pl

  A simple script for extracting the UMI from a Fastq read derived from Qiagen’s Small RNA library preparation kit. Typically, a 60 base or longer read is needed. Since small RNAs are typically under 30 bases, the sequence goes through the RNA insert, Qiagen adapter, and index code. This script searches for the Qiagen adapter sequence, allowing for up to two mismatches, and extracts the remaining sequence as the UMI.

Bam applications

These post-alignment applications are intended to work on Bam files for deduplication based on coordinates and UMI sequence. They work on files only (no streaming).

• bam_umi_dedup.pl

  A generic, multi-threaded, UMI-aware Bam de-duplication application. Alignments may be marked or removed based on coordinate, strand, and UMI sequence. UMI duplicates are selected based on highest quality scores (mapping and base quality). All single-end, proper paired-end, supplementary, and secondary alignments are processed, with caveats: supplementary, chimeric, secondary, and mate pairs on separate chromosomes are treated independently.

Deprecated applications

These are deprecated scripts, kept here for posterity, but superseded by other scripts. Generally do not use. The scripts can be found in the deprecated folder.

• SingleEndFastqBarcodeTagger.pl

  A simple script for incorporating a second Fastq read of UMIs into the first Fastq file, appending the UMI to the read name.

• qiaseq_bam_deduplication.pl

  A paired-end, UMI-aware Bam de-duplication application designed to work with the qiaseq fastq scripts above. UMI duplicates are selected for the highest quality sequence. Superseded by the generic bam_umi_dedup.pl.

• qiaseq_barcode_extractor.pl

  A simple script to extract the UMI code from the beginning of the second Fastq read (paired-end) derived from the Qiagen Qiaseq PCR-amplicon based library preparation kit. It will remove the UMI code and write it as a third Fastq file. This is designed to work as a Qiagen-specific pre-processor for using the USeq FastqBarcodeTagger, MatchMates, and Consensus applications for collapsing PCR duplicate reads into a single consensus read for re-alignment. See Workflows for examples.

• qiaseq_FastqBarcodeTagger.pl

  A simple script, similar to qiaseq_barcode_extractor.pl, to extract the UMI from Qiagen Qiaseq PCR-amplicon based library preparation and appending the UMI to the read name. Intended as a Qiagen-specific replacement processor for the USeq tool FastqBarcodeTagger and analogous to embedded_UMI_extractor.pl. Reads may then be aligned as normal and de-duplicated using the below scripts.