Applied Plant Genomics (GE31/MM12)

This repository contains materials and scripts used for Applied Plant Genomics.

Calculate Sequence Read Stats

This script calculates several statistics of a given FASTQ file: number of reads, number of sequenced nucleotides, average read length (mean), GC content, and N50 length of the reads. The script can handle uncompressed and compressed FASTQ files.

Usage:
  python3 FASTQ_stats3.py
 
Mandatory:
  --in   STR   FASTQ file

De novo Genome Sequence Assembly

Canu is an established tool for the generation of long read plant genome sequence assemblies. Alternatives are miniasm, Flye, and shasta.

Here is an explanation how to run Canu:

/vol/data/tools/canu-2.2/bin/canu \
-d /vol/data/members/<USER_NAME>/assembly/ \
-p <ASSEMBLY_NAME> \
genomeSize=1m \
-nanopore /vol/data/data/reads.fastq.gz \
minReadLength=10000 \
> /vol/data/members/<USER_NAME>/docu.txt \
2> /vol/data/members/<USER_NAME>/err.txt &

Calculate Assembly Statistics

This script calculates general assembly statistics and removes short contigs below a certain cutoff.

Usage:
  python3 contig_stats3.py
 
Mandatory:
  --input           STR    Assemblyfile
 		
Optional:
  --out              STR    Output folder
  --min_contig_len   INT   Minimal contig length [500]

Run AUGUSTUS for Gene Prediction

AUGUSTUS is one of the standard tools for the generation of ab initio gene predictions.

augustus \
--gff3=on  \
--UTR=on \
--uniqueGeneId=true \
--codingseq=on \
--species=arabidopsis \
ASSEMBLY \
> annotation.gff

Extract Peptide and Coding Sequences

There is an AUGUSTUS-associated perl script for the extraction of peptide and coding sequences called getAnnoFasta.pl. This script requires the genome sequence assembly file (FASTA) and the annotation file (GFF) as input.

getAnnoFasta.pl \
--seqfile=ASSEMBLY_FILE \
ANNOTATION_GFF_FILE

Add Functional Annotations to Predicted Genes

This script allows to assign functional annotation terms to predicted genes based on sequence similarity to previously characterized sequences.

Usage:
  python3 construct_anno3.py
 
Mandatory:
  --out OUTPUT_FOLDER \
  --in PREDICTED_PEPTIDE_FILE \
  --ref ath.pep.fasta \
  --anno ath.anno.txt

Long Read Mapping

minimap2 can be used to align long reads to a genome sequence.

/vol/data/tools/minimap2-2.24_x64-linux/minimap2 \
-ax map-ont --secondary=no -t 10 \
GENOMSE_ASSEMBLY_FASTA \
FASTQ_FILE \
> MAPPING_FILE

samtools view -Sb \
SAM_FILE \
> BAM_FILE

samtools sort -@ 10 \
-o OUTPUT_BAM_FILE \
INPUT_BAM_FILE

samtools index <BAM_FILE>

References

Pucker B, Holtgräwe D, Rosleff Sörensen T, Stracke R, Viehöver P, Weisshaar B (2016) A De Novo Genome Sequence Assembly of the Arabidopsis thaliana Accession Niederzenz-1 Displays Presence/Absence Variation and Strong Synteny. PLoS ONE 11(10): e0164321. doi:10.1371/journal.pone.0164321.

Pucker B, Holtgräwe D, Stadermann KB, Frey K, Huettel B, Reinhardt R, et al. (2019) A chromosome-level sequence assembly reveals the structure of the Arabidopsis thaliana Nd-1 genome and its gene set. PLoS ONE 14(5): e0216233. doi:10.1371/journal.pone.0216233.

Pucker, B., Kleinbölting, N. & Weisshaar, B. Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis. BMC Genomics 22, 599 (2021). doi:10.1186/s12864-021-07877-8.