Table of contents

• Authors
• Dependencies
• Description
• Algorithm
• Usage
• Output

Authors

• Myo T. Naung
• Somya Mehra

Dependencies

1. Nucleotide-Nucleotide BLAST 2.9.0+
2. R version 4.0 and above
3. automake/1.15

Description

Probe Maker has built as executable program using Make and it is intended for design of probes for Plasmodium falciparum for long-read sequencing technologies. It is comprised of three main steps - initial probe findings to conserved regions, nucleotide blast, and final probe selection. Based on the species would like to be designed, the following files (inside data directory) need to be changed accordingly.

1. pf3k.rds (species specific) (Plasmodium falciparum variant database based on Naung et al., 2022).
2. BLAST Database (access for Plasmodium falciparum (3D7), Plasmodium vivax (Pv01), and Human (Hg38))

Algorithm

As the hybridization takes place at 60°C based on STAR-seq protocol, we designed baits such that they had a melting temperature greater than this. Each probe is designed 500 nt apart. The algorithm designed the 120 bp probes to conserverd region based on database. Since the hybridization process between the probe and DNA fragment is tolerant to some mismatches, we permitted seven mismatches (i.e., 7 SNPs) out of 120 nt window in the design. GC content was however ignored from the design. Probe-Maker checked for potential cross hybridisation with human and other major malaria species using Blast. For each gene of interest, steps of the algorithm are as follow:

1. Finding 120-mer conserved regions and calculating melting temperature of each region
2. Removing regions that has potential cross hybridisation with human and other major malaria species using Blast.
3. Finding a set of the most suitable 120-mer probes to each gene with 500 bps apart.

Figure 1: Description of Probe-Maker algorithm

Usage

First of all, probe.txt file is required to be replaced by the informations of the gene that need to be used. The input information to the probe.txt has to be in the exact order as shown in the example (i.e. ref/reference.fasta, gene ID, chromosome ID, start coordinate, end coordinate, output directory). Comma (,) is required to seperate between each input information. The required reference gene in FastA format has be placed inside the ref folder.

After loading BLAST and R > 4.0 in the computation environment, Probe Maker can be downloaded (as zip file from Open Science Framework (OSF) repository) and executed as following:

cd Probe-Maker
make all

Each step of the software can be run separately given that the required input for each step has completed.

make help

Usage:
  make all                      #entire pipeline

Usage:
  make candidate                #for initial step

Usage:
  make blast                    #blastn but need outputs from candidate step

Usage:
  make final_probes             #final probe selection but need outputs from candidate and blast steps

Output

The final designed probe can be accessed via _final_probe.fasta inside specified output directory. In the cases that needs to spike in or requires to add additional probes, please consult with _blast_candidates.txt (regions that passed Blastn filter) and _probes_candidates.fasta (that has fasta sequences for every possible probe sequences) inside cache folder.

The example output shown here is for AMA1 gene.