Annotation Steps for Grasses

Some of the steps were performed following the steps given here: https://github.com/PeanutBase/BIND_annotation

Citation

This repository contains scripts used in the following publication:

Add a citation here when it's available.

Step 1: Process RNA-Seq Files

Run FastQC

Trim reads using Trim Galore and fastp

Step 2: Masking the genome

Cat both haplotypes of the genome together

Run EDTA and soft-masking

Step 3: Align Reads to the genome

Align (cleaned and trimmed) reads to genome using HISAT2

Step 4.A: De novo transcriptome assembly

Run StringTie to assemble transcripts

Step 4.B: Identify homologous genes

Use GeMoMa with protein evidence from related species and RNA-seq alignments generated with HISAT2

Step 4.C: Filter splice junctions

Portcullis -- used as input for Mikado

Step 5: Pick best transcript for each gene locus

Combine output from StringTie and GeMoMa

Run Mikado pick pipeline

Step 6: Filtering gene models

Use TEsorter to remove likely TE genes

Use eggnog-mapper to filter for genes with orthology assignment

Use gFACs to filter out genes with poor structure

Step 7: Rename gene models

Rename all of the genes according to their order along the genome

Step 8: Functional annotation

Run Entap

Download Databases

Run configuration

Run entap

Run NLR gene predictors

NLR-Annotator (genomic loci)

NLRtracker (NLR genes)

Get overlap between different software results

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Software Used

Software	Location
FastQC	https://github.com/s-andrews/FastQC
fastp	https://github.com/OpenGene/fastp
Trim Galore	https://github.com/FelixKrueger/TrimGalore
HISAT2	https://github.com/DaehwanKimLab/hisat2
EDTA	https://github.com/oushujun/EDTA
StringTie	https://github.com/gpertea/stringtie
GeMoMa	https://www.jstacs.de/index.php/GeMoMa
GMAP	http://research-pub.gene.com/gmap/
Portcullis	https://github.com/EI-CoreBioinformatics/portcullis
Transdecoder	https://github.com/TransDecoder/TransDecoder
Mikado	https://github.com/EI-CoreBioinformatics/mikado
TEsorter	https://github.com/zhangrengang/TEsorter
eggnog-mapper	https://github.com/eggnogdb/eggnog-mapper
gFACs	https://gitlab.com/PlantGenomicsLab/gFACs
EnTAP	https://gitlab.com/PlantGenomicsLab/EnTAP
DIAMOND	https://github.com/bbuchfink/diamond/
NLR-Annotator	https://github.com/steuernb/NLR-Annotator
NLRtracker	https://github.com/slt666666/NLRtracker
AGAT	https://github.com/NBISweden/AGAT
gffread	https://github.com/gpertea/gffread
bedtools	https://github.com/arq5x/bedtools2
samtools	https://github.com/samtools/samtools