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Run RNA-seq pipeline using the Telomere-2-Telomere (T2T) latest human genome

Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion–base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies (Nurk et al., Science, 2022 https://www.science.org/doi/10.1126/science.abj6987).

GRCh38 vs. T2T assemblies

• GRCh38:

  • Genome Reference Consortium Human Build 38 was released in December 2013.

  • 24 chromosomes (including the X and Y chromosomes) and 261 additional scaffolds that have not been assigned to a chromosome

  • Approximately 151 gaps in the primary sequence of GRCh38. These gaps are typically located in highly repetitive or hard-to-sequence regions such as centromeres, telomeres, and regions of segmental duplications.

• T2T:

  • The Telomere-to-Telomere (T2T) consortium released the T2T assembly in 2021 being the first truly gapless human genome assembly (T2T-CHM13), which further improved upon GRCh38 by closing these gaps. However, GRCh38 remains the reference genome widely used in genomics projects.

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Create the metadata file (samplesheet.csv):

Change to the data folder directory:

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cp /work/training/2024/rnaseq/scripts/create_samplesheet_nf-core_RNAseq_PEdata.sh $HOME/workshop/2024-2/session4_RNAseq/data/human

• Note: you could replace ‘$HOME/workshop/data’ with “.” A dot indicates ‘current directory’ and will copy the file to the directory where you are currently located

View the content of the script:

Example for Paired-End data (when ‘Read 1’ and ‘Read2’ are available) - Copy available script if working with PE data:

cat /work/training/2024/rnaseq/scripts/create_samplesheet_nf-core_RNAseq_SEdata.sh

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NOTE: modify ‘read1_extension’ and ‘read2_extension’ as appropriate for your data. For example: R1.fastq.gz, R2.fastq.gz or R1_001.fq.gz or , R2_001.fq.gz , etc

Prior running the “create_samplesheet” script, we need to know the path to the current (working directory) - run pwd and copy the path as we will use it in the subsequent block of code:

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We can use the nf-core/rnaseq pipeline to profile the expression of genes in a custom genome (e.g., T2T or any animal or plant genome) of your interest, as long as there is a reference genome (FASTA file) and genome annotation (GTF or GFF3).

To use your own genome assembly - you need 1) FASTA genome sequence and 2) GFF/GTF genome annotation file

--fasta my_custom_genome.fasta  # de novo assembled genome or genome not available as an igenomes reference
--gtf my_custom_genome.gtf      # genome annotatio showing the location of genes

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cd $HOME/workshop/2024-2/session4_RNAseq/runs/run4run3_RNAseq/_T2T

Copy and paste the code below to the terminal:

cp $HOME/workworkshop/training/2024-2/rnaseqsession4_RNAseq/data/human/samplesheet.csv $HOME/workshop/2024-2/session4_RNAseq/runs/run3_RNAseq_T2T
cp $HOME/workshop/2024-2/session4_RNAseq/scripts/launch_nf-core_RNAseq_pipeline_T2T.pbs $HOME/workshop/2024-2/session4_RNAseq/runs/run3_RNAseq_T2T

• Line 1: Copy the samplesheet.csv for pre-downloaded human samples file to the working directory

• Line 2: Copy the launch scrip to run expression profiling using the T2T genome

Print the content of the “launch_nf_core_RNAseq_T2T.pbs” script:

cat launch_nf_-core_RNAseq_pipeline_T2T.pbs

#!/bin/bash -l
#PBS -N nfRNAseq_T2T
#PBS -l select=1:ncpus=2:mem=4gb
#PBS -l walltime=48:00:00
#work on current directory
cd $PBS_O_WORKDIR
#load java and set up memory settings to run nextflow
module load java
export NXF_OPTS='-Xms1g -Xmx4g'
#run the RNAseq pipeline
nextflow run nf-core/rnaseq --input samplesheet.csv \
        --outdir results \
        -r 3.14.0 \
        --fasta /work/training/references/ncbi/T2T/GCF_009914755.1_T2T-CHM13v2.0_genomic.fna \
        --gtf /work/training/references/ncbi/T2T/GCF_009914755.1_T2T-CHM13v2.0_genomic.gtf \
        --remove_ribo_rna \
        -profile singularity \
        --aligner star_salmon \
        --extra_trimgalore_args "--quality 30 --clip_r1 10 --clip_r2 10 --three_prime_clip_r1 1 --three_prime_clip_r2 1 " \
        -resume

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Submit the job to the cluster

qsub launch_nf_-core_RNAseq_pipeline_T2T.pbs

Tip: Read the help information for Nextflow pipelines

Information on how to run a nextflow pipeline and additional available parameters can be provided on the pipeline website (i.e., https://nf-co.re/rnaseq/3.12.0/docs/usage/ ). You can also run the following command to get help information:

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Some pipelines may need file names, and others may want a CSV file with file names, the path to raw data files, and other information.

Public genomes

ENSEMBL publishes a range of genome assemblies and annotation files for a broad range of species. Look for species of interest at:

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