Molecular-Inversion-Probes

Author: Priya Lakra

Designing MIPs for non-human genes

This workflow will generate molecular inversion probes for non-human genes, for instance, bacterial, viral and fungal genes.

Requirements:

Python

MAFFT Aligner

Workflow:

1. Sequences for AMR genes were obtained from https://card.mcmaster.ca/ontology/ database.

2. Sequences were aligned using MAFFT.sh script.

3. Multiple sequence alignment was used to identify regions containing extensive gaps, and such regions were avoided in downstream analysis.

4. Design of molecular inversion probes (MIPs).

• The general format for the molecular inversion probes (MIPs) used here is a common NNN bp linker flanked by an extension arm of 18 to 20 bp and a ligation arm of 20 to 24 bp.
• The unique arms of each MIP target a specific 100 bp genomic region.
• Linker can be the NGS sequencing adaptors and sample-specific barcodes.
• MIPs for a given target region were chosen iteratively from the 5' to 3' end of the target region. Each successive MIP is chosen to satisfy the following criteria: 1) resides on the opposite strand as the previous MIP, 2) doesn’t overlap with previous MIP, 3) avoids gapped regions as obtained from MSA.
• Good probe features: lack polyNs (6) GC : 40-70 tm >= 60
• If no suitable MIPs can be picked, 2nd condition can be avoided.

run —help flag for inputs in pl_path_mips.py

Designing MIPs for human genes

MIPgen pipeline by Shendurlab

Dependencies: Bwa 0.7.17 samtools 1.11

1. Obtain MIPgen

   bash downloadmipgen.sh <path-to-store-mipgen-folder>
   

2. Generate index from Human reference genome file (GRCh38.primary_assembly.genome.fa)

    mkdir index
    bwa index -p GRCh38.primary_assembly.genome.fa  /data1/priya/new_data/reference/bwa/GRCh38.primary_assembly.genome.fa  
   

Keep the name of index and fasta file same

3. Running MIPgen

    input="location where MIPGEN is stored"
    cd $input
    ./mipgen -regions_to_scan /data1/priya/mipgen/MIPGEN/mipgen_practice/practice_genes.bed -project_name /data1/priya/mipgen/MIPGEN/tools/prac_design -min_capture_size 162 -max_capture_size 162 -bwa_genome_index /data1/priya/new_data/reference/GRCh38.primary_assembly.genome.fa