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MSSPE (Metagenomic Sequencing with Spiked Primer Enrichment) Design Software v1.0

Last updated 10/23/19 Charles Chiu

Overview

This is a shell script that takes as input a multiple-sequenced aligned (MSA) FASTA file of reference sequences and designs short primer sets (generally 11-17 nt) tiled across the entire sequence space, for use in the MSSPE protocol.

The MSSPE protocol has been published in Deng, et al., (2019) in the journal Nature Microbiology.

v1.0 - uses legacy BLASTALL (software used for the Deng, et al. 2019 paper)

This software has been tested in an Ubuntu 16.04.6 LTS Linux environment.

Main script: MSSPE-design.sh [OBFUSCATED]

Usage

MSSPE-design.sh <MSA FASTA file> <kmer size> <segment_window> <overlap> <selection_window> <evalue> <dG> <dS> <numcores>

*********************************************************************************************************************************
MSA FASTA file = multiple-sequenced aligned FASTA file
kmer size = kmer size (generally 11 - 17 nt)
segment_window = designated bp interval between forward and primer spiked primers (300-600 nt)
overlap = overlap in the tiled fragment windows (1/2 of the fragment window size, or 150-300 nt)
selection_window = size of window in bp from which forward and reverse primers will be selected (50 nt)
evalue = evalue for comparing forward and reverse primers by BLASTN (generally 0.1)
dG = "delta G", primer energy threshold cutoff in cals/mol (generally -9000)
dS = "delta S", cutoff for the number of minimum segments that need to be covered by each new primer (generally 0-10, depending
   on the number, diversity, and breadth of reference sequeces
*********************************************************************************************************************************

Software package dependencies:

  1. pyfasta (included with software package under MIT license

  2. NCBI BLAST 2.7.1+ package (requires blastn and makeblastdb)

  3. Python v2.7.12

  4. Perl v5.22.1

  5. ntthal from Primer3 package

Additional required scripts:

  1. annotateTm.py
  2. iterate_checkdimer.sh [OBFUSCATED]
  3. kmer_count_fasta_top50.pl

Please note that the key scripts "MSSPE-design.sh" and "iterate_checkdimer.sh" have been obfuscated. Please contact Dr. Charles Chiu, University of California, San Francisco (UCSF) at charles.chiu@ucsf.edu if you would like to make changes or improvements to the script.

******************************************************************************************************************************
Sample Test File:
1. ZIKV-96seqs.fasta

    To run with 8 cores, type "MSSPE-design.sh ZIKV-96seqs.fasta 13 500 250 50 0.1 2 -9000 8" from the command line.
******************************************************************************************************************************

Hardware & Software Requirements

Additional Software Dependencies

Required Scripts

Instructions for Running the MSSPE Design Software

  1. Generate a multiple-sequenced aligned (MSA) FASTA file (using "-" to denote gaps in the alignment). Available software tools include MAFFT, accessed October 2019, MUSCLE, accessed October 2019, and several others.
  2. Run the MSSPE-design.sh script:

MSSPE-design.sh <MSA FASTA file> <kmer size> <segment_window> <overlap> <selection_window> <evalue> <dG> <dS> <numcores>

*************************************************************************************
• MSA FASTA file = multiple-sequenced aligned FASTA file
• kmer size = kmer size (generally 11 - 17 nucleotides / nt)
• segment_window = designated bp interval between forward and primer spiked primers (300-600 nt)
• overlap = overlap in the tiled fragment windows (1/2 of the fragment window size, or 150-300 nt)
• selection_window = size of window in bp from which forward and reverse primers will be selected (50 nt)
• evalue = evalue for comparing forward and reverse primers by BLASTN (generally 0.1)
• dG = "delta G", primer energy threshold cutoff in cals/mol (generally -9000)
• dS = "delta S", cutoff for the number of minimum segments that need to be covered by each new primer (generally 0-10, depending on the number, diversity, and breadth of reference sequences
• numcores = number of available cores on your computer
*********************************************************************************************************************************

Test Run

  1. A sample test file named ZIKV-96seqs.fasta is provided, which generates MSSPE spiked primers based on 96 ZIKV reference genomes in the NCBI (National Center for Biotechnology Information) GenBank database

  2. Run the MSSPE-design.sh script from the command line with the following parameters (using 8 cores):

    MSSPE-design.sh ZIKV-96seqs.fasta 13 500 250 50 0.1 2 -9000 8

  3. The MSSPE ZIKV primer sequences in FASTA format can be found in

    ZIKV_96seqs.nospace.forward.13mers.fasta ZIKV_96seqs.nospace.reverse.13mers.fasta

    and summary statistics can be found in:

    summary_statistics_ZIKV-96seqs.13mers.txt