GTseq-ploidy

Modified version of the GTseq pipeline (Campbell et al. 2015) for polysomic polyploids, as described in Willis et al. (2020) Single nucleotide genotypes and ploidy estimates for ploidy variable species generated with massively parallel amplicon sequencing Authorea

Perl, bash, R, and python scripts are provided to run the GT-seq pipeline from fastq files containing sequences from GT-seq style amplicon-sequencing. While named for white sturgeon (Acipenser transmontanus), the ploidy variable polysomic species for which it was designed, it should be agnostic to species given the correct primer/probe sequence file.

Please note: while the genotyper perl script (GTseq_Genotyper_v3_Tetra.pl) initially records tetraploid genotypes, the probe (allele) counts are then used by an R script (called by Atr_GTseq_GenoCompile_ploidy_v1.sh) to infer ploidy and create genotypes corrected by that inferred ploidy.

Prerequisites for these scripts are the installation of perl, python, parallel (linux), and R (3.6; has NOT BEEN TESTED with 4!). Required R packages should be installed automatically with active internet connection. The scripts should be placed in the path (e.g. /usr/local/bin/). They have only been tested extensively on RHEL and come with NO WARRANTY.

An R script containing functions used in Willis et al. (2020) is also provided, ploidy_genotype_etc_from_genos.R. It is intended to be run piecemeal in e.g. Rstudio. Example genos input files containing 1) parents and offspring from a known cross containing ploidy variation and 2) samples of known but variable ploidy are provided. Genos files are an intermediate product of the GT-seq pipeline containing probe (allele) counts for each locus, one file per individual. The script is designed to be run with the folder containing the genos files as the working directory. This script is compatible with genos files created by earlier "tetra" versions of the pipeline (which assumed ploidy was 4N).

Please describe problems with the pipeline or R script in the Github Issues tab. Please note that we do not have sufficient resources to provide support for basic Linux, perl, bash, R issues that are dataset/user-specific.

AtrGTseq-PLOIDYv1 (325)

This code assumes that fastq files have been correctly demultiplexed prior to pipeline implementation. Genotype the samples using the following scripts. Make sure to verify the genotyping is complete before moving on. Use the command TOP and do not proceed until finished.

Navigate to the folder containing the fastq files, eg.

cd /data/GTseq_Libraries/NS_0000/AtrGTseqV2.0/Individuals/

Run the shell script which creates a command to count reads in each fastq

Assumes that GTseq_Genotyper_v3_Tetra.pl and Atr325_ProbeSeqs.csv are in the current working directory or in the path sh Atr325_GTseq_GenotyperShellGenerator.sh > NS_0000_Atr325_Genotyper.sh

Run the script just created which contains the commands; creates *.genos files

sh NS_0000_Atr325_Genotyper.sh

Replace 'Lxxxx' with the appropriate library name

make directory for the genos files mkdir Lxxxx_Genos/

Move the genos file to this directory

mv i*genos Lxxxx_Genos/

Change working directory to this genos directory

cd Lxxxx_Genos/

See options for the ploidy estimation and genotyping script

bash Atr_GTseq_GenoCompile_ploidy_v1.sh -h

Run the script (here with default options, but using multiple cores)

bash Atr_GTseq_GenoCompile_ploidy_v1.sh -p y

Make directory for the negative control genos

mkdir N_T_C/

Move negative control genos to that directory

mv *N_T_C*genos N_T_C/ cd N_T_C/

Create genotypes of negative controls (assumes tetraploid)

NTC_GTseq_GenoCompile_v3_Tetra.pl > Lxxxx_N_T_C.csv cd ..

Create summary files

GTseq_SummaryFigures_v3_Tetra.py
>> /data/GTseq_Libraries/NS_0000/AtrGTseqV2.0/Individuals/Lxxxx_Genos/
>> Lxxxx
convert *png Lxxxx.pdf
rm -f *png

References

Campbell, N. R., Harmon, S. A., & Narum, S. R. (2015). Genotyping-in-Thousands by sequencing (GT-seq): A cost effective SNP genotyping method based on custom amplicon sequencing. Molecular Ecology Resources, 15(4), 855–867. doi: 10.1111/1755-0998.12357

Willis, S.C., T. Delomas, B. Parker, D. Miller, P. Anders, S. Narum (2020) Single nucleotide genotypes and ploidy estimates for ploidy variable species generated with massively parallel amplicon sequencing. Authorea