Programme

Nadia Pinto

Filipe Pereira, Pedro Ferreira, Eran Elhaik, Henk Braig

Arrival: Sunday, 30 August

Day 1, 31 August, 09h – 17h

Non-Human Forensic Genetics: Methodologies, Applications, Exceptions, and Case Studies

Nádia Pinto, i3S-Institute for Research and Innovation in Health, University of Porto, Portugal, and Henk Braig, National University of San Juan (UNSJ), San Juan, Argentina

Foundations and scope

• Basic concepts in non-human forensic genetics

• Why non-human genetic material matters in forensic contexts

• When the field behaves like human forensic genetics and when it does not

Methodologies

• Sampling, extraction, quantification, and validation

• Marker types

• Software and laboratory workflow, including sequence curation and quality control

Interpretation

• Database-based identification and its limits

• Statistical evaluation of evidence

• Phylogenetic and evolutionary inference, including recombination and model choice

Applications

• Zoology, botany, microbiology, and food forensics

• Species identification, individualization, kinship, population assignment, outbreak tracing, and traceability

Exceptions and pitfalls

• Nonstandard or difficult cases

• Low-quality databases, poor population structure, degraded samples, mixtures, and validation limits

• Situations where the usual forensic assumptions do not hold

Case studies

Day 2, 01 September, 09h – 17h

Molecular Identification of Non-Human Biological Evidence: A Comprehensive Workflow (hands-on) by

Filipe Pereira, Department of Genetics and Biotechnology (DGB), University of Trás-os-Montes and Alto Douro, Portugal

• Basic concepts related to biological identifications using DNA

• Overview of DNA sequencing in the laboratory

• Introduction to software for DNA sequence analyses

• Installation and use of the Geneious software for DNA sequence management

• Sequence curation and quality control (electropherograms, formats)

• Brief overview of alignment of DNA sequences and phylogenetic inferences

• Database-based identifications (BLAST, BOLD): strengths and pitfalls

• Examples of real cases of using DNA for biological identifications

• Data interpretation and reporting

Day 3, 02 September, 09h – 17h

Introduction to Biological Sequence Databases Pedro G. Ferreira, Computer Sciences Department (hands-on), Faculty of Sciences of the University of Porto, Portugal

• Sequence data formats and metadata.

• Types of biological sequence databases (primary/non-curated and secondary/curated).

• How sequences are organized, annotated, and linked.

• Retrieving sequences efficiently.

• Database quality assessment and common pitfalls.

• Using sequence databases in non-human forensic genetics and metagenomics.

Introduction to Metagenomic Data Analysis: From Raw Sequencing Reads to Microbial Profiles (hands-on), Eran Elhaik, Ancient DNA Hub, Lund, Sweden

Introduction to Metagenomics and Microbiome Research

• § What metagenomics is and why it is used

• § Difference between shotgun sequencing and amplicon sequencing

• § Applications in ecology, health, and forensic science

Understanding Sequencing Data

• What sequencing reads are

• Structure of FASTQ files

• Paired-end sequencing and quality scores

Quality Control and Data Cleaning

• Why sequencing data must be cleaned

• Identifying sequencing errors and contaminants

• Read trimming and filtering

• Example tools: FastQC, MultiQC, fastp, Trimmomatic

Day 4, 03 September, 09h – 17h

Introduction to Metagenomic Data Analysis: From Raw Sequencing Reads to Microbial Profiles – continuation, Eran Elhaik

Preparing Reads for Analysis

• Handling paired-end reads

• Removing host DNA contamination

• Mapping reads to reference genomes

• Example tools: repair.sh, Bowtie2, Samtools

Microbial Identification and Classification

• Assigning sequencing reads to microbial taxa

• How reference databases are used

• Generating microbial abundance tables

• Example tools: Kraken2 and Bracken

Amplicon Analysis and Microbial Community Profiles

• Processing amplicon sequencing data

• Merging reads and removing chimeras

• Clustering sequences and generating OTU tables

• Example tools: PANDAseq and VSEARCH

Workshop’s challenge

Workshop Dinner

Departure: Friday, 4 September

Logistics

You must bring your own laptop!

Venue

The workshop will be held at i3S, organized with support from the i3S Advanced Training Unit.

i3S-Institute for Research and Innovation in Health, University of Porto

Rua Alfredo Allen, 208

4200-135 Porto, Portugal

GPS coordinates: 41º 10’ 30.008’’ N, 8º 36’ 12.488’’ W.