Ji Research Group In The Division of Oncology

OS-Seq primer probe design

Introduction

Oligonucleotide-Selective SEQuencing (OS-Seq) is an integrated automated targeted sequencing approach for high depth interrogation of high numbers of genomic regions-of-interest (ROI). In an automated cBot protocol, an Illumina flow cell is modified with target specific primer probe oligonucleotides, turning the flow cell surface into a target enrichment platform. Subsequently a sequencing library is hybridized against these target specific primer probes and enriched for ROI, after which the flow cell is prepared for sequencing (Figure 1). We developed a computational pipeline for optimized design of target specific primer probe oligonucleotides. This design pipeline has been demonstrated to give improved capture uniformity and capture rate, while vastly increasing the number of targeted bases.

OS-seq Figure1

Figure 1. (click for larger view) Figure 1: Overview of OS-Seq method.

REQUIREMENTS FOR OS-SEQ DESIGN PIPELINE

Dependencies

Data files

Script

User-created files


DIRECTIONS FOR RUNNING THE PIPELINE

Download files:

The OS-Seq_scripts.zip contains 2 required folders (lowercase):

The “inputs” folder should contain a tab delimited txt-file named “coordinates_to_extract.txt” with 4 columns (no header) :

The target regions will be increased in size by 500 bases to find optimal positions for the primer probes.

The “scripts” folder should contain the matlab script “os_seq_design_from_coordinates_v3.1.m”:

From within the scripts folder, run the following command to find optimally placed primer probes in the listed target regions:

nohup nice time matlab -nodisplay -nodesktop -nojvm -nosplash <os_seq_design_from_coordinates_v3.1.m>osseq.log 2>&1 &


OUTPUT

The “results_design” folder contains 2 tab-delimited files:

The "results_annot" contents are subsets of the kmer files for each target region to reduce memory usage and the scores folder in “results_design” lists the scores for each of these kmer positions and are summarized in the “output_by*” files.

Within the “scripts” folder a log file (“osseq.log”) is created for error messages.

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