nazeeefa / nanoswe

NanoSwe: Analysing nanopore (PromethION) data of Swedish genomes

NanoSwe: Analysing PromethION Sequencing Data of Swedish Genomes 🇸🇪

Introduction

NanoSwe is a preliminary analysis toolkit for experiments that involve sequencing data from ONT's PromethION device. It has also been used for other long-read SweGen data (e.g. PacBio).

Bioinformatics ToolKit

Purpose	Program
Quality Control	NanoPlot for QC and NanoComp
Mapping to the reference	Minimap2-2.14
Sorting, Indexing, and calculating statistics	Samtools 1.9
Subsampling	Sambamba 0.7.1
BAM QC Statistics	Qualimap 2.2.1
Structural Variant Calling	Sniffles 1.0.10
Data Extraction (VCF Files only)	bcftools 1.9
Finding intersection in genomic regions	Survivor 1.0.7
Evaluation of SVs	Survivor 1.0.7 and surpyvor: 0.5.0
Removing control DNA sequences	NanoLyse
Trimming Short Reads	BBMap/BBTools
Homology Detection	Blast 2.7.1+
Data Visualisation	R version 3.5.3. See the scripts directory for information on libraries/packages used.

Nanopore Data: Post-Sequencing

Example tree structure of nanopore sequencing data files

├── /basecalled/<sample>/<flowcell>/
│   ├── fastq_0.fastq
│   ├── fastq_850.fastq
│   ├── sequencing_summary_0.txt
│   ├── sequencing_summary_850.txt
│   └── reads (1)
│       ├── 0 (2)
│       │   ├── file_read_1_ch_90_strand.fast5
│       │   ├── file_read_41_ch_40_strand2.fast5
│       │   └── file_read_300_ch_40_strand2.fast5
│       └── 850
│           ├── file_read_1000_ch_200_strand.fast5
│           ├── file_read_9000_ch_100_strand.fast5
│           └── file_read_95000_ch_1000_strand2.fast5
└── /bin/

(1) Each folder contains ~8000 fast5 files
(2) fast5 file named e.g. PCT0001_YYYYMMDD_0001A20B002222C_{flowcell}_sequencing_run_{library_full_name}__read_{number}_ch_{number}_strand.fast5)

Nanopore Data: Post-Tidying

Example tree structure of data organisation

├── /basecalled/<sample>/<flowcell>/
│   ├── FASTQ_files
│   │   ├── fastq_0.fastq
│   │   └── fastq_850.fastq
│   ├── sequencing_summary
│   │   ├── sequencing_summary_0.txt
│   │   └── sequencing_summary_850.txt
│   ├── reads *
│   │   ├── 0 *
│   │   │   ├── file_read_1_ch_90_strand.fast5
│   │   │   ├── file_read_41_ch_40_strand2.fast5
│   │   │   └── file_read_300_ch_40_strand2.fast5
│   │   └── 850
│   │       ├── file_read_1000_ch_200_strand.fast5
│   │       ├── file_read_9000_ch_100_strand.fast5
│   │       └── file_read_95000_ch_1000_strand2.fast5
│   └── <sample>_analysis
│       ├── reference_genome.fna
|       ├── reference_genome.fna.fai
│       ├── Snakefile
│       ├── /bam_files/
│       ├── /vcf_files/
│       └── /logs/
└── /bin/

Sub-folder content

./scRipts - R scripts created for visulisation of long read data.
commands.md - Tool commands used for different analyses.

Data Sources

• SweGen: a whole-genome data resource of genetic variability in a cross-section of the Swedish population
• De novo Assembly of Two Swedish Genomes Reveals Missing Segments from the Human GRCh38 Reference and Improves Variant Calling of Population-Scale Sequencing Data.
• Multi-platform discovery of haplotype-resolved structural variation in human genomes

Recommended Readings

• Which human reference genome to use?
• Structural variants identified by Oxford Nanopore PromethION sequencing of the human genome
• The thesis
• Evaluating nanopore sequencing data processing pipelines for structural variation identification

Recommended Pipeline(s)

• nfcore/nanoseq
• Nano-snakemake
• Long-Read Data Analysis Tutorial

Citation

If you plan to use repository as a guide, simply and kindly mention the link https://github.com/Nazeeefa/NanoSwe for acknowledgment. To cite our publication, you can cite it as as shown below otherwise visit citeas.org to choose a different format. Thank you.

AMA Style

Fatima N, Petri A, Gyllensten U, Feuk L, Ameur A. Evaluation of Single-Molecule Sequencing Technologies for Structural Variant Detection in Two Swedish Human Genomes. Genes. 2020; 11(12):1444.

Chicago Style

Fatima, Nazeefa; Petri, Anna; Gyllensten, Ulf; Feuk, Lars; Ameur, Adam. 2020. "Evaluation of Single-Molecule Sequencing Technologies for Structural Variant Detection in Two Swedish Human Genomes." Genes 11, no. 12: 1444.