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Before logging on
- If off campus - Download the VPN
- Connect
Option 1: Terminal Application
- Open a terminal with one of these:
- Mac: Terminal (native, usually found in Applications -> Utilities) or iTerm2
- Mac or Windows: mobaxterm - Use portable edition
- Mac or Windows: Visual Studio Code
- Log into sphinx or isaac head node:
ssh username@sphinx.ag.utk.edu
or
ssh username@login.isaac.utk.edu
Option 2 (Later): ISAAC On Demand
- Navigate in your web browser to login.isaac.utk.edu
- Click on Clusters -> ISAAC Shell Access
Complete. Great job, everyone! See course information here: Module 1
This module will last for 7 class periods. The grading will be as follows:
- 80% Project-based test
- 20% In class discussion participation (2)
- Illumina Slides - Short Read Platforms, Fastq Format, Error Profiles, Intro to lab data
- Lab: Module 2.1 - project set up, conda, fastqc, multiqc
- Homework: Ask an AI assistant of your choice to write you a complete short-read SNP calling pipeline for a haploid fungal genome, starting from paired FASTQ and ending at a filtered VCF. Do not edit it. Do not run it. Submit the raw output verbatim, plus a note recording which model you used, the exact prompt, and the date. Points for doing this are part of your final test.
- Lab: Start on software download so it can run while we talk about read mapping
- Slides: Read mapping and SAM/BAM/CRAM
- Reading
- Nielson 2013 Genotype and SNP calling from next-generation sequencing data - this is for discussion
- Danecek 2021 Twelve years of SAMtools and BCFtools - this is a good reference, not needed for discussion
- Discussion: Day 10 discussion seeds
- Lab: Module 2.2 - fastp
- Slides: File formats (vcf/bcf)
- Lab: Module 2.4 bcftools and vcf filtering
- Optional: Fix conda speed issue (hopefully)
- Homework:
- Download Jbrowse2: https://jbrowse.org/jb2/download/
- Download IGV: https://igv.org/doc/desktop/#DownloadPage/
- Reading for all: Li H 2014. Toward better understanding of artifacts in variant calling from high-coverage sequencing data. - not going to be discussed, but interesting for understanding when and why snp calling fails
- HW Readings: Select the article most relevant to your area of study and be ready to discuss on Thursday with peers who selected the same paper. Each group will report on the most important points to the rest of the class.
- Marees et al., 2018: A tutorial on conducting genome-wide association studies: Quality control and statistical analysis
- Hoban et al., 2016: Finding the Genomic Basis of Local Adaptation: Pitfalls, Practical Solutions, and Future Directions
- Leache and Oakes, 2017: The Utility of Single Nucleotide Polymorphism (SNP) Data in Phylogenetics
- Sun & Li 2026: Advances in haplotype phasing and genotype imputation
- Discussion Seeds:
- What is the key insight you took away from this paper?
- Are there any areas you wish the authors had expanded further that left you with additional questions?
- Was there anything you didn't understand about this paper that we can clarify as a class?
- Share a bit about your own project with your group. Did you learn anything that you can apply to your own work, either technically or conceptually?
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Discussion
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Slides: VCF Filtering (Continued)
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Try to find
- Clean SNP - Consistent alt allele, balanced strands, good MAPQ
- Indel-adjacent misalignment - Ragged pileup of "SNPs" beside an indel
- Repeat region - MAPQ 0 reads, spurious calls?
- Strand bias - Alt allele on one strand only
- Homopolymer - Length-variable indel calls
- Slides: https://docs.google.com/presentation/d/10CeNgG8AQG81MJdBoaLDNawafleOTdqpWTqCmpP0FDI/edit?slide=id.p#slide=id.p
- Lab: Module 2.6 SnpEff, SNP Ordination, and Combining VCFs
- Assign test - see below
Test 2 help in class
Test 2 help in class
- Test 2 Explanation & Rubric: Test 2: A celebration of your new SNP calling expertise
- Test 2 Template to Copy into your own GitHub Repo: Test 2 Wiki template raw
- Example of Rendered Test 2 Template: Test 2 Wiki template rendered
- October 13th - Optional deadline to turn in work for Steps 1-6, from fastqc to evaluating your SNP sets against the ground truth set. Zane and I will help you figure out if you have any issues to fix prior to writing the reports.
- October 20th - Final due date
This module will last for 7 class periods. The grading will be as follows:
- 60% Project-based test
- 40% In class discussion participation (4)
- ISAAC intro - scaling up!
- Guest speaker Dr. Ryan Kuster.
- HW Readings:
- Marx 2023 Method of the year: long-read sequencing
- Mahmoud 2025 A Hitchhiker's Guide to long-read genomic analysis
- Discussion - Discussion prompts for long read sequencing overview papers
- Lab - Long Read QC and mapping - with slurm!
- Lecture - SV Slides
- Benchmarking papers mentioned in lecture: Liu 2024-human,Zhang 2026-plants
- CuteSV papers: Jiang 2020, Jiang 2025
- Great infographic from the Broad using a sweater as an analogy for SV categories
- Lab - Long Read SV calling and JBrowse viewing
- Papers - pick one. We will get in groups by paper. Come prepared to discus how the methods accomplish SV detection and how SV data is used for biological inference
- Yano et al (2025) The genomic landscape of gene-level structural variations in Japanese and global soybean Glycine max cultivars Nature Genetics
- Weissensteiner et al. (2020) Discovery and population genomics of structural variation in a songbird genus
- Schloissnig et al. (2025) Structural variation in 1,019 diverse humans based on long-read sequencing Nature.
- Worakitchanon et al. (2024) Comprehensive analysis of Mycobacterium tuberculosis genomes reveals genetic variations in bacterial virulence Cell Host & Microbe
- Discussion
- Lab - SVs impacting genes
- Lecture
- Lab
- Discussion
- Lab
- Go vote!
- Discussion
- Lab
- TEST 3 in class help
Pangenomes or RNASeq or other???