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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.

T2T genome

The latest T2T human genome and annotation has been downloaded from NCBI:

https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/009/914/755/GCF_009914755.1_T2T-CHM13v2.0/

You can access this genome at: /work/training/references/ncbi/T2T

Check available files:

Code Block
ls -l /work/training/references/ncbi/T2T/
Code Block
GCF_009914755.1_T2T-CHM13v2.0_assembly_report.txt
GCF_009914755.1_T2T-CHM13v2.0_genomic.fna
GCF_009914755.1_T2T-CHM13v2.0_genomic.gff
GCF_009914755.1_T2T-CHM13v2.0_genomic.gtf
GCF_009914755.1_T2T-CHM13v2.0_protein.faa
GCF_009914755.1_T2T-CHM13v2.0_rna.fna

Print the assembly report:

Code Block
cat /work/training/references/ncbi/T2T/GCF_009914755.1_T2T-CHM13v2.0_assembly_report.txt

...

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.

T2T genome

The latest T2T human genome and annotation has been downloaded from NCBI:

https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/009/914/755/GCF_009914755.1_T2T-CHM13v2.0/

You can access this genome at: /work/training/references/ncbi/T2T

Check available files:

Code Block
ls -l /work/training/references/ncbi/T2T/
Code Block
GCF_009914755.1_T2T-CHM13v2.0_assembly_report.txt
GCF_009914755.1_T2T-CHM13v2.0_genomic.fna
GCF_009914755.1_T2T-CHM13v2.0_genomic.gff
GCF_009914755.1_T2T-CHM13v2.0_genomic.gtf
GCF_009914755.1_T2T-CHM13v2.0_protein.faa
GCF_009914755.1_T2T-CHM13v2.0_rna.fna

Print the assembly report:

Code Block
cat /work/training/references/ncbi/T2T/GCF_009914755.1_T2T-CHM13v2.0_assembly_report.txt
Code Block
# Assembly name:  T2T-CHM13v2.0
# Description:    T2T CHM13v2.0 Telomere-to-Telomere assembly of the CHM13 cell line, with chrY from NA24385
# Organism name:  Homo sapiens (human)
# Taxid:          9606
# BioSample:      SAMN03255769
# BioProject:     PRJNA559484
# Submitter:      T2T Consortium
# Date:           2022-01-24
# Assembly type:  haploid
# Release type:   major
# Assembly level: Complete Genome
# Genome representation: full
# Expected final version: no
# Genome coverage: 30x
# GenBank assembly accession: GCA_009914755.4
# RefSeq assembly accession: GCF_009914755.1
# RefSeq assembly and GenBank assemblies identical: no
#
## Assembly-Units:
## GenBank Unit Accession       RefSeq Unit Accession   Assembly-Unit name
## GCA_009914825.4      GCF_009914825.1 Primary Assembly
## GCA_009914855.2              non-nuclear
#
# Ordered by chromosome/plasmid; the chromosomes/plasmids are followed by
# unlocalized scaffolds.
# Unplaced scaffolds are listed at the end.
# RefSeq is equal or derived from GenBank object.
#
# Sequence-Name Sequence-Role   Assigned-Molecule       Assigned-Molecule-Location/Type GenBank-Accn    Relationship    RefSeq-Accn     Assembly-Unit   Sequence-Length    UCSC-style-name
1       assembled-molecule      21       Chromosome      CP068276CP068277.2      =       NC_060926060925.1     Primary Assembly        242696752248387328       chr2chr1
32       assembled-molecule      32       Chromosome      CP068275CP068276.2      =       NC_060927060926.1     Primary Assembly        201105948242696752       chr3chr2
43       assembled-molecule      43       Chromosome      CP068274CP068275.2      =       NC_060928060927.1     Primary Assembly        193574945201105948       chr4chr3
54       assembled-molecule      54       Chromosome      CP068273CP068274.2      =       NC_060929060928.1     Primary Assembly        182045439193574945       chr5chr4
65       assembled-molecule      65       Chromosome      CP068272CP068273.2      =       NC_060930060929.1     Primary Assembly        172126628182045439       chr6chr5
76       assembled-molecule      76       Chromosome      CP068271CP068272.2      =       NC_060931060930.1     Primary Assembly        160567428172126628       chr7chr6
87       assembled-molecule      87       Chromosome      CP068270CP068271.2      =       NC_060932060931.1     Primary Assembly        146259331160567428       chr8chr7
98       assembled-molecule      98       Chromosome      CP068269CP068270.2      =       NC_060933060932.1     Primary Assembly        150617247146259331       chr9chr8
9 10      assembled-molecule      9 10      Chromosome      CP068268CP068269.2      =       NC_060934060933.1     Primary Assembly        134758134150617247       chr10chr9
1110      assembled-molecule      1110      Chromosome      CP068267CP068268.2      =       NC_060935060934.1     Primary Assembly        135127769134758134       chr11chr10
1211      assembled-molecule      1211      Chromosome      CP068266CP068267.2      =       NC_060936060935.1     Primary Assembly        133324548135127769       chr12chr11
1312      assembled-molecule      1312      Chromosome      CP068265CP068266.2      =       NC_060937060936.1     Primary Assembly        113566686133324548       chr13chr12
1413      assembled-molecule      1413      Chromosome      CP068264CP068265.2      =       NC_060938060937.1     Primary Assembly        101161492113566686       chr14chr13
1514      assembled-molecule      1514      Chromosome      CP068263CP068264.2      =       NC_060939060938.1     Primary Assembly        99753195101161492       chr14
chr15
1615      assembled-molecule      1615      Chromosome      CP068262CP068263.2      =       NC_060940060939.1     Primary Assembly        9633037499753195        chr16chr15
1716      assembled-molecule      1716      Chromosome      CP068261CP068262.2      =       NC_060941060940.1     Primary Assembly        8427689796330374        chr17chr16
1817      assembled-molecule      1817      Chromosome      CP068260CP068261.2      =       NC_060942060941.1     Primary Assembly        8054253884276897        chr18chr17
1918      assembled-molecule      1918      Chromosome      CP068259CP068260.2      =       NC_060943060942.1     Primary Assembly        6170736480542538        chr19chr18
2019      assembled-molecule      2019      Chromosome      CP068258CP068259.2      =       NC_060944060943.1     Primary Assembly        6621025561707364        chr20chr19
2120      assembled-molecule      2120      Chromosome      CP068257CP068258.2      =       NC_060945060944.1     Primary Assembly        4509068266210255        chr21chr20
2221      assembled-molecule      2221      Chromosome      CP068256CP068257.2      =       NC_060946060945.1     Primary Assembly        5132492645090682        chr22chr21
X 22      assembled-molecule      X22       Chromosome      CP068255CP068256.2      =       NC_060947060946.1     Primary Assembly        51324926 154259566       chrXchr22
YX       assembled-molecule      YX       Chromosome      CP086569CP068255.2      =       NC_060948060947.1     Primary Assembly        154259566       chrX
Y      62460029 assembled-molecule      Y       Chromosome      CP086569.2      =       NC_060948.1    chrY MTPrimary Assembly     assembled-molecule      MT      Mitochondrion   CP068254.1      <>      na      non-nuclear     16569   chrM    62460029        chrY
MT      assembled-molecule      MT      Mitochondrion   CP068254.1      <>      na      non-nuclear     16569   chrM

Create the metadata file (samplesheet.csv):

Change to the data folder directory:

Code Block
cd $HOME/workshop/2024-2/session4_RNAseq/data/human

Copy the bash script to the working folder

Code Block
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:

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

...

Let’s generate the metadata file by running the following command:

Code Block
sh create_samplesheet_nf-core_RNAseq_PEdata.sh $HOME/workshop/2024-2/session4_RNAseq/data/human

Check the newly created samplesheet.csv file:

Code Block
cat samplesheet.cvs

Run RNAseq pipeline using a custom genome

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

Code Block
--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

...

Code Block
cp /work/training/2024/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:

...

Code Block
qsub launch_nf_core_RNAseq_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:

...

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:

...