Bioinformatic pipeline for analysing variations in long-reads reconstructed human genomes

Angelo Sante Varvara; Ilenia Urso; Sharon Natasha Cox; Graziano Pesole

Sep 30, 2024

Version 1

Bioinformatic pipeline for analysing variations in long-reads reconstructed human genomes V.1

DOI

dx.doi.org/10.17504/protocols.io.36wgqnoz3gk5/v1

Angelo Sante Varvara¹,
Ilenia Urso²,
Sharon Natasha Cox¹,
Graziano Pesole^1,2

¹Department of Biosciences, Biotechnology and Environment, University of Bari Aldo Moro, Bari, Italy;
²Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies, Consiglio Nazionale delle Ricerche, Bari, Italy

Angelo Sante Varvara

University of Bari

DOI: https://dx.doi.org/10.17504/protocols.io.36wgqnoz3gk5/v1

Protocol Citation: Angelo Sante Varvara, Ilenia Urso, Sharon Natasha Cox, Graziano Pesole 2024. Bioinformatic pipeline for analysing variations in long-reads reconstructed human genomes. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqnoz3gk5/v1

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: July 08, 2024

Last Modified: September 30, 2024

Protocol Integer ID: 102990

Keywords: Bioinformatics, ONT, Nanopore, variants, human, variations, reads, indels, SNV, SV, alignment, phasing, dorado, clair3, whatshap, modkit, modified bases, variant calling, IGV, whole exome sequencing, use of either whole exome sequencing, clinical genomic, sequencing technology, whole genome sequencing, read sequencing, throughput sequencing, genome, bioinformatic pipeline, oxford nanopore technology, sequencing, genomic region, nanopore, unprecedented insights into the genetic basi, read, effective diagnostic tool for patient, effective diagnostic tool, genetic basi

Abstract

Recent advances in sequencing technologies have paved the way for personalized medicine and clinical genomics, providing unprecedented insights into the genetic basis of human health and disease. High-throughput sequencing is now recognized as an effective diagnostic tool for patients affected by rare diseases but the use of either Whole Exome Sequencing (WES) or Whole Genome Sequencing (WGS) technologies has shown to be uninformative in many cases. On the contrary, the recent introduction of long-read technologies can resolve genomic regions inaccessible to short-read sequencing, but a reliable benchmark assessment is missing. 

Following a long and intense benchmarking activity, here we present our bioinformatic pipeline based on Oxford Nanopore Technology (ONT) long reads aimed at shading lights to the stages of alignment, SNVs, indels, and SV calling, and modified bases analysis.

Attachments

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Troubleshooting

Before start

#Versions of tools used in this pipeline:

- Dorado 0.7.2
- samtools 1.19.2
- NanoStat 1.6.0
- pycoQC 2.5.2
- Clair3 1.0.9
- Clair3-Nova 0.3.0
- bcftools 1.19
- sniffles2 2.2
- sniffles2_plots 0.2.0
- SvAnna 1.0.4
- whatshap 2.1
- modkit 0.2.2

Please kindly note that whilst running Dorado without a GPU is technically possible, it is strongly inadvisable as basecalling will be much slower when running purely on CPU.
Dorado is heavily optimized for Nvidia A100 (ampere) and H100 (hopper) GPUs and will deliver maximum performance on nodes containing these GPUs.

Basecalling and alignment

11h

Basecalling raw data in pod5 format with Dorado basecalling.

Dorado is a high-performance open-source basecaller for Oxford Nanopore reads.
First, let's download the latest models for our needs with Dorado download.
Command
Downloading Dorado models
Downloading Dorado models
dorado download --model dna_r10.4.1_e8.2_400bps_sup@v5.0.0

dorado download --model dna_r10.4.1_e8.2_400bps_sup@v5.0.0_5mCG_5hmCG@v1
Then, we can run Dorado basecaller on our raw data in pod5. 
If your raw data are in fast5 format, it is recommended to convert them in pod5 with pod5 convert fast5 tool to reach optimal performances.
For our test, we are going to use human Genome assembly GRCh38 as a reference, calling both 5mC and 5hmC modified bases, using the most updated and accurate models.
Command
Basecalling with Dorado
Basecalling with Dorado
dorado basecaller dna_r10.4.1_e8.2_400bps_sup@v5.0.0 /sample/pod5/ --min-qscore 10 --recursive --modified-bases-models dna_r10.4.1_e8.2_400bps_sup@v5.0.0_5mCG_5hmCG@v1 
--reference hg38.fa > calls.bam
Following the basecalling step, create a sequencing summary with Dorado summary, it outputs a tab-separated file with read-level sequencing information from the BAM file generated during basecalling.
Command
Running Dorado summary
Running Dorado summary
dorado summary calls.bam > sequencing_summary_dorado.txt

Sort and index the resulting alignment with samtools.

It's time to sort the alignment in bam format with samtools sort.
Command
Sorting alignment
Sorting alignment
samtools sort -@ 16 calls.bam -o calls_sorted.bam
Indexing the sorted bam is mandatory for further steps.
Command
Indexing alignment
Indexing alignment
samtools index -@ 16 calls_sorted.bam

Alignment statistics

Create alignment statistics with NanoStat, samtools, and pycoQC.

NanoStat highlights alignment information as, for instance, N50 or read length and read quality.
Command
Running NanoStat
Running NanoStat
NanoStat --summary sequencing_summary_dorado.txt -t 12 > Nanostats.txt
Samtools utilities give us insights on coverage and mapping values.
Command
Running samtools stats and samtools coverage
Running samtools stats and samtools coverage
samtools stats -@ 16 calls_sorted.bam > samtools_stats.txt

samtools coverage calls_sorted.bam > samtools_coverage.txt
PycoQC produces an html format file with tons of charts regarding basecalling progress and outcomes.
Command
Running pycoQC
Running pycoQC
pycoQC -f sequencing_summary_dorado.txt -o report_dorado.html

Variant calling

2h 35m

SNV and indels calling with Clair3.

Variant calling is performed with Clair3 and we strongly advise these parameters, with the same model path used for the basecalling stage.
Command
Running clair3
Running clair3
run_clair3.sh --bam_fn="calls_sorted.bam" --ref_fn="hg38.fa" --threads=16 --platform="ont" --include_all_ctgs --model_path="dna_r10.4.1_e8.2_400bps_sup@v5.0.0" --output="clair3"

Polishing calls with bcftools.

This step depends on your alignment statistics. We usually advise removing all "lowQual" calls as well as variants with depth below 5 (or 10 if your coverage is above 30x).
Command
Filtering vcf
Filtering vcf
bcftools view merge_output.vcf.gz -i 'FILTER="PASS" & FMT/DP>5’ --threads 8 --output filtered_1.vcf.gz
Furthermore, bcftools norm command will split all multi-allelic variants into multiple rows and furtherly check calls with the reference throwing out redundancies or errors, producing a vcf file with improved accuracy.
Command
Running bcftools norm
Running bcftools norm
bcftools norm -m-any --check-ref -w -f hg38.fa filtered_1.vcf.gz -o filtered_2.vcf.gz
Lastly, it can probably be helpful to add further info to the final vcf file, especially in pathogenic studies. Annovar and SnpEff are both powerful tools that can fill variant calls with annotations, as those from ClinVar, Gnomad and dbSNP.

10m

SV calling with sniffles2.

We strongly advise using the bed files found in sniffles GitHub repository to achieve better performances while calling structural variants in repetitive regions.
Command
Running sniffles2
Running sniffles2
sniffles --input calls_sorted.bam --vcf sniffles.vcf --snf sniffles.snf --reference hg38.fa --minsvlen 50 --output-rnames --threads 16 --tandem-repeats human_GRCh38_no_alt_analysis_set.trf.bed
Calls can be subsequently filtered and polished depending on our needs with bcftools.

10m

Additionally, sniffles2 developers offer a python program that generates a wide range of plots for single and multi-sample VCF files, such as SV size & type distribution, and genotype frequency.
Command
Creating plots
Creating plots
python3 -m sniffles2_plot -i sniffles.vcf -o sniffles2_plots

SV annotation and prioritization with SvAnna.

Structural variants need to be annotated as well as, in case of pathogenic analysis, prioritized. We benchmarked several tools for this purpose, but we determined SvAnna to be the most suitable for our goals, taking as inputs either sniffles2 vcf and our HPOs. 
The output in html is specifically useful in order to have a first look at the most valuable structural variants in terms of pathogenic relevance.
Command
Running SvAnna
Running SvAnna
java -jar svanna-cli-1.0.4.jar prioritize -d /svanna-cli-1.0.4/svanna-data --output-format html,vcf --vcf sniffles.vcf --phenotype-term HP:0001250 --phenotype-term HP:0001249 --phenotype-term HP:0000256 --n-threads 8 --out-dir /svanna-cli-1.0.4/output

10m

Phasing

3h 35m

Phasing heterozygous variants with whatshap phase.

This tool allows phasing variants (called via clair3), annotating the info directly in our vcf file.
Command
Running whatshap phase
Running whatshap phase
whatshap phase -o phased.vcf.gz --reference hg38.fa --ignore-read-groups merge_output.vcf.gz calls_sorted.bam
Please note that also structural variants can be phased with whatshap phase in the same way.

30m

Phasing assessment with whatshap stats.

Assessing the amount of phased heterozygous variants is crucial, and we can achieve this with whatshap stats. It will output various statistics which can vary a bit depending on the alignment quality (coverage, N50, etc), but we can safely conclude that a percentage above 90% is expected in 30X covered genomes.
Command
Running whatshap stats
Running whatshap stats
whatshap stats --tsv=whatshap.tsv --gtf=whatshap.gtf phased.vcf.gz > stats_whatshap.txt

Tagging reads by haplotype with whatshap haplotag.

Quite often it is interesting to show the reads as well as the variants. For that, we can run the whatshap haplotag subcommand on the phased VCF file. It tags each read in a BAM file with HP:i:1 or HP:i:2 depending on which haplotype it belongs to, and also adds a PS tag that describes in which haplotype block the read is.
The command below creates a BAM file 'haplotagged.bam' with the tagged reads, which you can open in IGV, in order to visualize either haplotype assignment and modified bases.
Command
Running whatshap haplotag
Running whatshap haplotag
whatshap haplotag -o haplotagged.bam --reference hg38.fa phased.vcf.gz calls_sorted.bam

Tips for trios

In case we had a trio of samples (child, parent1, and parent2) the above stages of variant calling and phasing can be completed with a single command thanks to the Clair3-Nova tool. With few lines of code, we would be able to perform SNVs and indels calling, phasing heterozygous variants, and haplotagging input bam files with haplotype infos, the two latter steps ran via whatshap if we specify the tool path.
If needed, Clair3-Nova can output also gvcfs files.
Command
Running Clair3-Nova
Running Clair3-Nova
run_clair3_nova.sh \
  --bam_fn_c=child_sorted.bam \
  --bam_fn_p1=P1_sorted.bam \
  --bam_fn_p2=P2_sorted.bam \
  --output=clair3_nova \
  --ref_fn=hg38.fa \
  --threads=12 \
  --model_path_clair3=r1041_e82_400bps_sup_v430 \
  --model_path_clair3_nova=r1041_e82_400bps_sup_nova \
  --gvcf \
  --include_all_ctgs \
  --whatshap=/usr/bin/whatshap \
  --enable_output_phasing \
  --enable_output_haplotagging
 

12h

Modified bases

Modified bases calling and stats with modkit.

Modified bases can be called with modkit directly from the modBAM file, depending on the type of model chosen during the basecalling stage. Modkit can effectively count the following modified bases: 5mC, 5hmC, 6mA. It outputs a bedMethyl file, a table with the counts of base modifications from every sequencing read over each reference genomic position. No reference sequence is required (hence we strongly advise using it) and for user convenience, the counting process can be modulated using several additional transforms and filters, for instance the most basic of these is to report only counts from reference CpG dinucleotides.
Command
Running modkit pileup
Running modkit pileup
modkit pileup calls_sorted.bam pileup_m.bed --ref hg38.fa --log-filepath pileup.log -t 16
It is also possible to produce a summary to have an overview of the mod tags that are present in a BAM and get basic statistics. The default output is a totals table (designated by '#' lines) and a modification calls table. We ran the command with --no-sampling flag to count modified bases throughout the genome but if you prefer, you can choose a specific region or a specific number of reads to be randomly picked.
Command
Running modkit summary
Running modkit summary
modkit summary calls_sorted.bam --no-sampling -t 16
Furthermore, modkit offers the possibility to perform differential methylation scoring. This can be done using two subcommands: modkit dmr pair to compare regions in a pair of samples (for example, tumor and normal, or control and experiment), or modkit dmr multi to compare regions between all pairs of samples (for example, a trio sample set ). Before performing this step, the input data (i.e., bedMethyl files created in the previous step) must be compressed with bgzip and indexed with tabix. Additionally, you must specify a BED file containing the specific regions to be compared, such as CpG Islands in hg38. This BED file should contain 4 columns: chrom, chromStart, chromEnd, and CpGname. Keeping the name column is optional and we can also specify the modified base in which we are interested in, for example, for CpG islands we can specify "--base C". 
Command
Differential methylation scoring for a pair of samples
new command name
modkit dmr pair -a control.bed.gz --index-a control.bed.gz.tbi -b  case.bed.gz --index-b case.bed.gz.tbi -o differential_case_control.bed -r CpG.bed --ref hg38.fa --base C --threads 16 --log-filepath file.log
The modkit dmr multi command performs pairwise comparisons across all regions specified in the regions BED file for more than two samples. The data preparation is the same as described in the previous section, applied to each sample. An example command for a trio is the following: 
Command
Differential methylation scoring on all pairs of samples
new command name
modkit dmr multi -s father.bed.gz norm1 --index father.bed.gz.tbi norm1 -s mother.bed.gz norm2 --index mother.bed.gz.tbi norm2 -s child.bed.gz child1 --index child.bed.gz.tbi child1 -o out_path --regions-bed CpG.bed --ref hg38.fa --base C -t 32 --log-filepath file.log
The latest version of modkit introduces the option "--segments", which allows modkit dmr to dynamically identify and generate regions of differential methylation during the analysis using a hidden Markov model, instead of relying on pre-defined regions. This can provide more flexible and potentially more accurate identification of methylation patterns in your data
Command
Differential methylation using a Hidden Markov Model with --segments option
new command name
modkit dmr pair --segment segmentation.bed  -a control.bed.gz --index-a control.bed.gz.tbi -b case.bed.gz --index-b case.bed.gz.tbi -o out_path --ref hg38.fa --base C -t 32 --log-filepath file.log

Further analysis

Further analyses are expected to be performed according to the specific aim of your study. 

Public workspaceBioinformatic pipeline for analysing variations in long-reads reconstructed human genomes V.1

Bioinformatic pipeline for analysing variations in long-reads reconstructed human genomes V.1