UMI Scripts - smallRNA_pe_umi_extractor

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smallRNA_pe_umi_extractor

A script to extract the Unique Molecular Index (UMI) from the second read of a short-read (50 bp) paired-end Qiagen small RNA library. The structure of a typical small RNA paired-end read is as follows:

Since the small RNA insert may be of variable length, the full UMI sequence (12 bp by default) may not be reliably present within the full read length of R1, in which case extracting it from R2 becomes necessary.

The Qiagen adapter is identified in R2 (allowing for mismatches as necessary), the preceding UMI is extracted, and the UMI appended to the read name. The Qiagen adapter is also searched for and removed from R1 if present, allowing for mismatches and deletions as necessary.

Only one Fastq file, R1, is written out. Reads with adapter sequence in both R2 and R1 is always written to output. Reads with the adapter identified only in R2 is written to output or optionally to a second file (option --suspect). Reads where the adapter sequence is not found in either R2 or R1 may only be written to an optional file (option --fail). No UMI code is appended to failed reads.

Sequencing primers are not searched for, but normally should not be necessary except for long insertions (suspect reads).

The UMI is appended to the read name as “:NNNNNNNNNNNN”. After alignment, the bam file may be de-duplicated bam_umi_dedup.pl.

Usage:

smallRNA_pe_umi_extractor.pl --out <basename> --f1 <read_1> --f2 <read_2>

smallRNA_pe_umi_extractor.pl <read_1> <read_2>

Options:

-1 |--f1 <file>      First read in Fastq, may be gzipped
-2 |--f2 <file>      Second Fastq read, expected to contain the 
                       barcode. May be gzipped. Both files may also
                       be simply appended to the command line.
-o |--out <file>     Specify output fastq filename. Default is STDOUT.
                       GZip compression is fully supported.
-s |--suspect <file> Specify optional filename for suspect fastq reads
                       where the adapter is only found in Read2.
                       Default is to include in primary output.
-f |--fail <file>    Specify optional filename for failed fastq reads 
                       where the adapter cannot be found in either read.
                       Default is to not include in primary output.
-a |--adapt <ATGC>   The Qiagen adapter sequence to look for in Read2.   
                       Default is 'ATTGATGGTGCCTACAGTT'.
-l |--len <integer>  Length of the Unique Molecular Index. Default 12.
-h |--help           This help