Equus caballus Project

Aim

Assess sequence polymorphism in five horse genes of interest by comparing amplicon seq data of healthy and unhealthy horses.

Genes of interest:

Gene Name	Ensembl	NCBI Gene ID
Proglucagon (GCG)	ENSECAG00000005660	100051551
Melanocortin 2 receptor (MC2R)	ENSECAG00000003841	100057018
Melanocortin 4 receptor (MC4R)	ENSECAG00000001712	100050469
Proopiomelanocortin (POMC)	ENSECAG00000016388	100071524
Adiponectin (ADIPOQ)	ENSECAG00000002962	100059500

Proglucagon (GCG) gene - ENSECAG00000005660 has two gene splicing variants:

Name	Transcript ID	bp	Protein	Biotype	UniProt Match	Flags

Name	Transcript ID	bp	Protein	Biotype	UniProt Match	Flags
GCG-201	ENSECAT00000066569.1	708	232aa	Protein coding	A0A5F5PSR3	-
GCG-202	ENSECAT00000005849.3	705	180aa	Protein coding	F7ABP1	-

>EquCab3.0_Glucagon_GCG000001|GCG-201 cds:protein_coding
ATGAAAAGCATCTACTTTGTGGCCGGACTGTTTGTAATGCTGGTACAAAGCAGCTGGCAACGTTCGCTGCAGGACACAGAGGAGACATCCAGATCGTTCCCAGCTCCCCAGACAGACCCACTCAGTGATCCGGATCAGATGAATGAAGATAAGCGCCATTCTCAGGGCACGTTCACCAGTGACTACAGCAAGTATCTGGACTCCAGGCGTGCCCAGGATTTCGTGCAGTGGTTGATGAACACCAAGAGGAACAAGAATAACATTGCCAAACGTCATGATGAATTTGAGAGACATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGAAGGCGAGATTTCCCAGAAGAAGTCACCATCGTTGAAGAACTCCGCCGCAGACATGCCGATGGTTCCTTCTCTGATGAGATGAACACAGTTCTTGATAATCTTGCCACCCGGGACTTTATAAACTGGTTGCTTCAGACCAAAATTACTGACAGAGTCAATAGAATACCTGAAAGAGACTGCAACAGAGTAGACTCCCATAATGAAGAGGGCATCTTCAGAGGTGAAGGGGAGCCCAAGTGTAACAGCTTTTCAAGTTCCCTCTCTTCAGTGAGGATCATAAGAGGCACTCCATTCAAGGGGAAGTGTGCAATCTGA
>EquCab3.0_Glucagon_GCG_202000001|GCG-202 cds:protein_coding
ATGAAAAGCATCTACTTTGTGGCCGGACTGTTTGTAATGCTGGTACAAAGCAGCTGGCAACGTTCGCTGCAGGACACAGAGGAGACATCCAGATCGTTCCCAGCTCCCCAGACAGACCCACTCAGTGATCCGGATCAGATGAATGAAGATAAGCGCCATTCTCAGGGCACGTTCACCAGTGACTACAGCAAGTATCTGGACTCCAGGCGTGCCCAGGATTTCGTGCAGTGGTTGATGAACACCAAGAGGAACAAGAATAACATTGCCAAACGTCATGATGAATTTGAGAGACATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGAAGGCGAGATTTCCCAGAAGAAGTCACCATCGTTGAAGAACTCCGCCGCAGACATGCCGATGGTTCCTTCTCTGATGAGATGAACACAGTTCTTGATAATCTTGCCACCCGGGACTTTATAAACTGGTTGCTTCAGACCAAAATTACTGACAGGAAGTAA

Melanocortin 2 receptor (MC2R)

- ENSECAG00000003841 single splice variant:

Name	Transcript ID	bp	Protein	Biotype	UniProt Match	Flags

Name	Transcript ID	bp	Protein	Biotype	UniProt Match	Flags
MC2R-201	ENSECAT00000003809.3	891	297aa	Protein coding	F6Y3T7	-

>EquCab3.0_Glucagon_MC2R_201000001|MC2R-201 cds:protein_coding
ATGAAGCACATTGTCAATCTATATGAAAACATCAATGATACAGCAAGAAATAATTCAGACTGTCCTCTTGTGGTTTTGCCAGAAGAGATATTCTTCACAATATCCATCATTGGGGTTTTGGAGAATCTGATGATCCTTCTGGCTGTGATCAAGAATAAGAATCTCCAGTCACCAATGTACTTTTTCATTTGCAGCTTGGCCATTTCTGATATGTTGGGCAGCCTATATAAGATCCTGGAAAATATCCTGATCATGTTCAGAAACACAGGTTATCTCAAGCCTCGTAGCAATTTTGAAACCACAGCCGATGACATCATTGACTCTCTGTTCATCCTCTCCCTACTTGGGTCCATTTTCAGCCTGTCTGTGATCGCCGTTGACCGCTACATCACAATCTTCCATGCTCTGCAGTACCACAGCATTGTGACCATGCACCGTGCCATTGTTGTCCTGATAGTCATCTGGACGTGCTGCCTGGGCAGCGGCATCGCCATGGTGATCTTCTCCCATCACATCCCCACAGTGATCACCTTCACCTCGCTGTTCCCTCTCATGTTGGTCTTTATCCTATGCCTCTATGTGCACATGTTCTTGCTGGCCCGTTCCCATGCCAGGAAGATCTCAACCCTCTCTAGAGGCAACATGAAAGGGGCCATCACGCTGACCATCCTGCTCGGGGTGTTCATCTTCTGCTGGGCCCCTTTTGTCCTTCATGTCCTCTTAATGACATTCTGCCCAAATAACCCTTACTGTGTCTGCTACATGTCCCTCTTCCAGGTGAATGGCATGTTGATCATGTGCAATGCAGTCATCGACCCTTTTATATATGCCTTCCGGAACCCAGAGCTCAGGGAGGCATTCAAAAAGATGATCTTCTGCAACAGTTACCAG

Melanocortin 4 receptor (MC4R)

- ENSECAG00000001712 has a single splice variant:

Name	Transcript ID	bp	Protein	Biotype	UniProt Match	Flags

Name	Transcript ID	bp	Protein	Biotype	UniProt Match	Flags
MC4R-201	ENSECAT00000001527.3	1002	333aa	Protein coding	F6TMM6	-

>EquCab3.0_Glucagon_MC4R_201000001|MC4R-201 cds:protein_coding
AGGATGGACTCTACTCACCGCCATGGAATGCACACTTCTCTCCACTTCTGGAACCGCAGCACCTACGGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGGAGAATATTCTGGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGTAGCCTGGCTGTGGCTGACATGTTGGTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTGTTAAACAGTACAGATACGGACGCACAGAGTTTCACGGTGAATATTGATAATGTCATTGACTCAGTGATCTGTAGCTCCTTACTTGCATCAATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTATTTTACTATCTTTTATGCTCTCCAGTATCATAACATTATGACGGTTAAGCGGGTCGGGATCATCATAAGTTGCATCTGGGCAGCTTGCACGGTTTCGGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTCATGGCCAGACTTCACATTAAGAGGATCGCTGTCCTCCCAGGCACTGGCACCATCCGCCAAGGTGCCAACATGAAGGGGGCGATCACCTTGACCATATTGATTGGAGTCTTTGTGGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCCCAGAATCCATACTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGTGTAATTCCATCATCGATCCTCTGATCTATGCACTCCGGAGCCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTACCCTCTGGGAGGCCTTTGTGATTTGTCTAGCAGATACTAA

Proopiomelanocortin (POMC)

- ENSECAG00000016388 has three splice variants:

Name	Transcript ID	bp	Protein	Biotype	UniProt Match	Flags
POMC-201	ENSECAT00000064027.2	1323	267aa		A0A3Q2IBW0	-
				Protein coding
POMC-202	ENSECAT00000069674.1	1320	253aa		A0A5F5PNG1	-
				Protein coding
POMC-203	ENSECAT00000017172.3	1131	332aa		F6W8H2	-
				Protein coding

Adiponectin (ADIPOQ)

- ENSECAG00000002962

Name	Transcript ID	bp	Protein	Biotype	UniProt Match	Flags

Name	Transcript ID	bp	Protein	Biotype	UniProt Match	Flags
ADIPOQ-201	ENSECAT00000002827.3	1264	301aa	Protein coding	F7DZE7	-

ConsGenome pipeline: Creating a conda environment

See this tutorial for additional information: https://conda.io/projects/conda/en/latest/user-guide/tasks/manage-environments.html

The ConsGenome workflow requires the following tools specified in an ‘environment.yml’ file:

Creating a conda environment called ‘ConsGenome’

Activate the environment → this enables to use of the above tools. NOTE: prior to running netxflow need to activate the ConsGenome environment.

Deactivate the environment

Nextflow - ConsGenome pipeline

This is a QUT eResearch developed pipeline for 1) the de novo assembly of reference sequences (i.e., amplicon seq regions, metagenomics sequences, etc); 2) map and deduce a reference consensus genomics/transcript sequence based on HTS data collected for a given sample, and 3) identify nucleotide sequence polymorphism.

To run the pipeline prepare the following:

index.csv - a file describing the sample ID, the path to read1 and read2 if applicable
nextflow.config - a file to specify parameter options such as the genome/transcriptome/amplicon reference to use to map reads and predict a consensus sequence
launch.pbs - PBS Pro script to submit the ConsGenome job to the HPC cluster

Example index.csv file:

Example nextflow.config file:

Launch.pbs script: