UMI Scripts - embeded_UMI_extractor

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embeded_UMI_extractor

A script to pull out an UMI embedded within a sequence read fastq file.

Unique Molecular Indexes (UMIs) are short, random nucleotide sequences that can help to distinguish independent DNA molecules with the same sequence composition. By associating the UMI to a nucleotide molecule at the beginning of library preparation, identical sequence reads can be distinguished between unique, biologically-derived molecules and PCR-derived or sequencing artifact duplicates.

UMIs are expected to be at the beginning of the sequence read. The UMI barcode may or may not be followed by a short fixed sequence precdeding the sequence read. Either single-end or paired-end fastq files may be processed. Paired-end files may have an UMI in either one or both files. If a fixed sequence is provided, it must be found for the read to be written to output; paired-end UMI containing files must have both found.

UMIs can be merged into sequence read Fastq files in three ways:

  1. SAM attribute in Fastq comment Inserted as standard SAM attribute tags RX and QX in the read header. This is compatible with e.g. BWA, Bowties2 aligners. DEFAULT.

  2. SAM attribute in unaligned Bam format Exported as an unaligned SAM/BAM format with RX and QX attribute tags. This is compatible with e.g. Bowtie2, STAR, BWA, and Novoalign. Use the --bam option.

  3. Append to name Appended to the read name as “:UMI_sequence”. This is compatible with all aligners but requires special software to utilize. Non-standard. Use the --name option.

Alignments can be de-duplicated utilizing the UMI codes with external software. See bam_umi_dedup.pl in this software package, or Picard ‘UmiAwareMarkDuplicatesWithMateCigar’ as possibilities.

External utilities may be required for execution, inlcuding pigz and/or gzip, and samtools. These are searched for in your environment PATH.

Version: 3.01

Usage

embedded_UMI_extractor.pl -l 8 -f GTAGTA *.fastq.gz 

embedded_UMI_extractor.pl -1 file1.fastq.gz -2 file2.fastq.gz \
--bam --out unaligned.bam --length 8 -f GTAGTA -F ACGACG

Options

Input:

-1 --read1 <file>       First fastq read
-2 --read2 <file>       Second fastq read, optional
-u --umi <integer>      Read containing UMI: 1, 2, or 12 (default 1 or 12)

Output:

-o --out <file>         Path to output (input basename)
                          use 'stdout' for (interleaved) piping
-b --bam                Write output as unaligned BAM format
                          or SAM format if stdout or samtools unavailable 
-n --name               Append UMI to read name instead of SAM tag RX

UMI Options:

-l --length  <integer>  Length of UMI at beginning of read1
-L --length2 <integer>  Length of UMI at beginning of read2 (if different)
-f --fixed   <text>     Fixed sequence after UMI for read1 (optional) 
-F --fixed2  <text>     Fixed sequence after UMI for read2 (optional, if different)

Other:

--samtools <path>       Path to samtools (/usr/local/bin/samtools)
-h --help               Show full description and help