Dinh:COMPUTATIONAL/umi procedure
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UMI Procedure[edit]
- UMIs are assumed to be the first N bases in read 1 ALWAYS.
- Reads with UMIs are processed by getUMI.pl first. If the reads are PE, make sure that the PE reads are in order.
For each read or pairs: record r1_name, r1_sequence, and r1_qualities record r2_name, r2_sequence, and r2_qualities assign umi = first N bases of r1_sequence append umi to r1_name and r2_name: umi_r1_name, umi_r2_name (first field separated by "_") remove first N bases from r1_sequence remove first N quality scores from r1_qualities print umi_r1_name, trimmed_r1_sequence, and trimmed_r1_qualities to *R1.fq file print umi_r2_name, r2_sequence, and r2_qualities to *R2.fq file
- Afterwards, reads can be processed by adaptor trimming and mapping to get the sam file.
- SAM files are processed for clonal removal by sam_UMI_filter_Poisson.pl
Sort SAM files by mapping positions. Calculate the number of possible umi, (m) For each position mapped with more than 1 read mapped: extract umi from each read count how many times each umi are observed count how many unique umi are observed (k) calculate Po (the probability of a umi not being used) from the relations: k = m(1-Po) Use Po to calculate the absolute number of molecules (n) with the Poisson relation: Po = e^(-n/m) Since we know the absolute number of molecules (n), then: if number of reads is less than n, we allow all the reads. if number of reads is greater than n: start with allowing 1 read per umi increase number of reads allowed until number of reads allowed is equal to n. print allowed reads to new SAM output file. move on to next position.
- Output SAM file can be converted to BAM for further downstream processing.
- Further notes from last year on this procedure: http://genome-tech.ucsd.edu/LabNotes/index.php/Dinh/Dinh_2014/NOTES/2014-1-4