NARASIMHA Primer Designer

Introduction

A program to generate primers for the NARASIMHA Assay for Targeted RNA Sequencing to identify Chimeric Gene Fusions: https://www.nature.com/articles/s41408-020-0313-6

1. Prerequisites

python=3.6
pandas=1.0.1
biopython
bedtools=2.29.1
primer3 (libprimer3 release 2.5.0)
isPcr
fpdf

or create and activate environment in scripts directory

        $ conda env update -f ./scripts/environment.yaml  
        $ conda activate narasimha-primers

2. Creating Input file

a. BED file

Create a 6 column BED file with the follwing values in tab delimited columns. chromosome, start-coordinate end-coordinate, unique gene/exon name (without spaces), Score(default 1 for all), strand(+ or -) Create separate BED files for plus and minus strand genes

   EXAMPLE
     chr9  133710452  133710912  ABL1_EX1  1  +
     chr9  133729450  133729624  ABL1_EX2  1  +

OR ### b. FASTA/multi-FASTA file Alternatively, a fasta file can be provided as input with cDNA sequence of the gene of interest.

3. RUN

python3 ./script/narasimha.py -i -g <path to reference genome (.fasta)>

python3 ./script/narasimha_plus.py -i <path to BED file with + strand genes> -g <path to reference genome (.fasta)> -l <length of region for primer design> 
python3 ./script/narasimha_minus.py -i <path to BED file with - strand genes> -g <path to reference genome (.fasta)> -l <length of region for primer design>
EXAMPLE:
./narasimha_plus.py -i lung_panel_v3_format_plus.bed -g /home/reference_genomes/hg19_ref_index/hg19.fasta -l 65
./narasimha_minus.py -i lung_panel_v3_format_minus.bed -g /home/reference_genomes/hg19_ref_index/hg19.fasta -l 65

4. Output

A "primer_out_*" directory will be created as output. The narasimha_minus.py creates primer_out_minus as output directory, narasimha_plus.py creates primer_out_plus as output directory.

The round1 and round2 primers are present in the "final" directory present inside the above folders.

The final list of primers for both rounds can be found in "all_primers.csv" in the "final" directory.

If no primers are generated, this may be due to short length of sequences.

For sequences less than 50 bases, it is recomended that the sequence of the previous exon also be taken and a fasta file be created. Run the program with the fasta file as input.

5. Visualization

The scripts pdf_writer_minus.py and pdf_writer_plus.py can be used to visulaize the output in pdf format.
The inputs required are sequence.fasta, round1primers.csv and round2primers.csv. These files can be found in the primer_out_plus(minus) directory from the above step.

The round1 and round2 primers are present in the "final" directory present inside the primer_out_plus(minus) folders.

sequence.fasta file is present inside the "temp" directory present inside the primer_out_plus(minus) folders. Run the scripts using the following format:

    python3 ./pdf_writer_plus.py sequences.fasta round1primers.csv round2primers.csv
    python3 ./pdf_writer_minus.py sequences.fasta round1primers.csv round2primers.csv