Skip to content

README.md

Latest commit

 

History

History
79 lines (60 loc) · 6.68 KB

README.md

File metadata and controls

79 lines (60 loc) · 6.68 KB
Raw

Ancient DNA analysis pipeline

This workflow performs an ancient DNA (aDNA) based analysis similar to the one in the nf-core/eager workflow. nf-core/eager is a bioinformatics best-practice processing pipeline for genomic NGS sequencing data, with a focus on ancient DNA data. It is ideal for the (palaeo)genomic analysis of humans, animals, plants, microbes and even microbiomes.

The pipeline processes the sequencing-read input provided to the workflow together with a reference genome and optional supporting reference data. It aligns reads and performs extensive general NGS and aDNA-specific quality control on the results.

Required & Optional Inputs

To run this workflow successfully, you need to provide the following input datasets and parameters:

  • Input reads : Input single-end FASTQ reads for the sample.
  • Reference genome : Reference genome sequence in FASTA format. This is essential for read mapping and variant calling.
  • Choose Mapper : Switch to select the alignment tool. Choose between BWA and Bowtie2.
  • Hap Map ChrX reference : HapMap dataset required for X-chromosome contamination estimation in ANGSD.
  • Input Mitochondrial Chromosome Name : The exact header name of the mitochondrial chromosome in your reference FASTA file (e.g., MT, chrM, rCRS).
  • Kraken2 database directory : The database directory required for Kraken2 taxonomic classification.
  • Optional BED file for Sex.DetERRmine : An optional BED file containing specific genomic coordinates to restrict the Sex.DetERRmine analysis. Leave empty for standard whole-genome human analysis, or provide targeted regions to enable gender estimation for non-human organisms.
  • ANGSD region parameter : The specific genomic region to restrict the ANGSD analysis (typically 'X' for human male nuclear contamination estimation).

Workflow Steps

By default the pipeline currently performs the following:

1. Preprocessing and Quality Control

  • Quality Control: Evaluates read quality before and after trimming (FastQC)
  • Adapter Trimming: Removes adapter sequences (AdapterRemoval)

2. Read Mapping and Processing

  • Alignment: Maps reads to the provided reference genome conditionally using either (BWA) or (Bowtie2) based on user selection
  • Filtering and Statistics: Separates unmapped reads and calculates alignment statistics (Samtools View and Flagstat)
  • Duplicate Removal: Detects and marks PCR duplicates (Picard MarkDuplicates)
  • Alignment Quality: Generates detailed BAM quality metrics (QualiMap BamQC)
  • Library Complexity: Estimates library complexity (Preseq)

3. Ancient DNA (aDNA) Analysis

  • Damage Profiling: Visualizes aDNA-specific C-to-T damage patterns (mapDamage)
  • Endogenous Content: Calculates the proportion of endogenous (target) DNA in the sample (EndorSpy)
  • (Optional) Contamination: Estimates nuclear X-chromosome contamination conditionally if HapMap data is provided (ANGSD X-Contamination)

4. Biological Information

  • Sex Determination: Determines biological sex based on relative chromosome coverage ratio. This step adapts conditionally whether an optional BED file is provided (Sex.DetERRmine)
  • Mt/Nuc Ratio: Calculates the ratio of mitochondrial reads to nuclear reads utilizing the specified mitochondrial chromosome name (MtNucRatioCalculator)

5. Genotyping

  • Variant Analysis: Performs variant calling to generate VCF files (FreeBayes)
  • Variant Statistics: Calculates statistics for the generated variants (Bcftools stats)

6. Metagenomic Screening (For Unmapped Reads)

  • Read Extraction: Extracts unmapped reads for microbial analysis (Picard SamToFastq)
  • Quality Filter: Filters low-complexity sequences (BBTools BBduk)
  • Taxonomic Classification: Performs microbiome/taxonomic screening on the filtered unmapped reads (Kraken2)

7. Reporting

  • Summary Report: Aggregates logs and statistics from all these tools into a single interactive HTML report (MultiQC)

Workflow Outputs

Upon successful execution, the workflow explicitly provides the following final files for analysis:

  • MultiQC aggregated workflow summary report : An interactive HTML report aggregating QC and analysis logs from all tools.
  • QualiMap BamQC general alignment quality metrics report : A detailed HTML report containing mapping quality metrics, GC content, and coverage statistics.
  • mapDamage Visualisation : Visual plots displaying the characteristic C-to-T deamination patterns at the ends of ancient DNA reads.
  • Kraken2 taxonomic classification and microbial screening report : A tabular report showing the taxonomic classification of unmapped reads.
  • EndorSpy endogenous DNA authentication report : A JSON file containing the calculated endogenous DNA percentage.
  • Sex.DetERRmine (Without BED) report of chromosomal gender estimation : A JSON file containing biological sex metrics for human-genome alignments.
  • Sex.DetERRmine (With BED) report of chromosomal gender estimation : A JSON file containing biological sex metrics for targeted capture regions.
  • Mitochondrial to nuclear DNA ratio calculation report : A JSON file containing the calculated ratio between mitochondrial and nuclear reads.
  • ANGSD report of nuclear contamination estimation : A tabular text file detailing the estimates of nuclear X-chromosome contamination.
  • Bcftools variant calling summary statistics report : A text file containing comprehensive summary statistics for the called variants (VCF).
  • Fully post-processed mapping results : The final deduplicated and filtered alignment BAM file.
  • Freebayes raw genomic variant calls : The raw VCF file generated from variant analysis.

Testing Data

To ensure the workflow functions correctly, it was validated using the following datasets and databases:

  • Primary Test Data : A downsampled single-end FASTQ dataset NIST7035_TAAGGCGA_L001_R1_001_10MB.fastq.gz optimized for rapid workflow testing and validation.
  • Primary Reference Genome : The hs37d5_chr21-MT.fa.gz file was utilized as the primary reference genome sequence.
  • X-Chromosome Contamination Reference : The HapMap ChrX dataset was provided as the initial reference for the estimation of X-chromosome contamination using the ANGSD tool.
  • Taxonomic Classification Database : The Minikraken v2 database was utilized to perform taxonomic classification via Kraken2.