SMOPINseqTools

Overview

Small molecule-protein interaction sequencing (SMOPIN-seq) is a high-throughput sequencing technology that efficiently detects small molecule-protein associations in vitro. Here, we distribute SMOPINseqTools, a standardized data processing pipeline to identify small molecule-protein associations from fastq files of SMOPIN-seq experiments.

Workflow

1. Raw read pairs from the PRIM-seq experiment are present in .fastq files.
2. The reads are first searched for small molecule library barcode with with 1 base editing tolerance.
3. The reads with library barcode are then searched for 3 cycles of moledule barcodes with 1 base editing tolerance.
4. The read-ends with library and molecule barcodes are assigned as the small-molecule-end reads. The corresponding other ends eassigned as the protein-end reads.
5. Cutadpt is applied to remove 3' linker sequences and 5' adapter sequences from the protein-end reads.
6. Fastp is then applied to the adapter-trimmed protein-end reads to remove low-quality reads whose mean quality is lower than Q20 and too short reads whose length is shorter than 20 bp.
7. The remaining protein-end reads are mapped to transcriptome with BWA with default parameters.
8. The mapped protein-end reads are output in .bed file with aligned genes and transcriptome alignment information.
9. The protein-end reads are paired with mapped small-molecule reads by read ids. Deduplications are then performed based on UMIs.
10. The kept small-molecule-protein pairs are output as small molecule-protein associations in SmoProteinAssociations.csv.

Software Requirements

• Cutadapt (2.5 or later)
• fastp (0.22.0 or later)
• bwa (0.7.17-r1188 or later)
• samtools (1.6 or later)
• bedtools (2.30.0 or later)
• Python 3.4 or later, the following python libraries are required:
  
  • sys
  • collections
  • cigar
  • glob
  • scipy
  • datetime

Additional files required

** White lists of library and molecule barcodes**
You will need a directory of lists of library barcodes and molecule barcodes in the first step to sort raw read pairs into molecule-end reads and protein-end reads, in the form of BB-Codon Map_HGP0001-OpenDEL0001.txt

BWA Index of the transcriptome to be aligned
You will need to download or build the bwa index of the target trancriptome for PROPERseqTools to use. Here we provide the compressed bwa index built from RefSeq GRCh38 transcriptome

Transcript, gene and gene type dictionary file
You will also need a dictionary file that contains the information of transcript ids to their corresponding gene names/gene ids and corresponding gene types in a csv format with the first column being transcript ids, the second column being gene names and the third column being gene types. Here we provide an example dictionary file for RefSeq GRCh38 genome

Usage

Installation

1. Clone the current github repository to your local machine. For example
   git clone https://github.com/Zhong-Lab-UCSD/SMOPINseqTools
2. Add the following path of the cloned directory to your .bashrc file
   export PATH=$PATH:/home/path/to/SMOPINseqTools/bin

To excute PROPERseqTools, run

SMOPINseqTools -a /path/to/read1.fastq
               -b /path/to/read2.fastq
               -i /path/to/bwaIndex/transcriptome.fa
               -l /path/to/smallMolecule/mapDirectory/
               -o /path/to/outputDir/
               -g /path/to/refSeq_tx_gene_type.csv
           

Required parameters

-a     |String, Path to read1 fastq file, fastq.gz also supported
-b     |String, Path to read2 fastq file, fastq.gz also supported
-o     |String, Path to output directory
-i     |String, Path to bwa index of the target transcriptome
-g     |String, Path to transcirpt, gene and gene type dictionary file
-l     |String, Path to the directory of lists of library and molecule barcodes

Other parameters

-j     |String, Job ID to be prepended to the output files and directories, optional, default=PROPERseq"
-t     |Int, Number of working threads, default=2
-r     |Char, (T or F), removal of intermediate and processed fastq files or not, default=T
-h     |Print usage message"