COURSE DESCRIPTION
Sequencing-based research has become the dominant investigative practice within the biological sciences. Single-Molecule & Spatial Sequencing: Methods & Applications responds to this development by providing a hands-on and in-depth introduction to sequencing for first-year PhD and MD-PhD students, from Sanger to third-generation technologies. Utilizing an alternating lecture-lab schedule, students are introduced to fundamental basic principles of DNA & RNA science, progressing to cutting-edge library preparation and sequencing analysis techniques with Illumina and Nanopore technologies. Students will have the opportunity to perform direct-RNA sequencing samples on research samples and experience first-hand the ethical implications of this data. Relevant concepts in biology and computer science will be addressed.
COURSE SCHEDULE
I. What is Single-Molecule Sequencing? (April 1st)
- Sequencing technologies: Illumina, MGI, Sanger, Singular [no single molecules] (Mason)
- Course Overview & Sequencing technologies: Nanopore & PacBio; public data [single molecule] (Mason)
- Single-molecule DNA-base calling accuracy vs. variability; sources of signal noise (Mason)
II. Lab #1: Dry Lab Practice & Single-Molecule Sequencing Formats (April 8th)
- Data wrangling and visualization in Python (Park / Nelson / Students)
- File Formats for Spatial and Single Cell Data: h5ad, RDS (Park)
- Single-molecule basecalling: pod5, slow5, blow5, SAM, BAM (Gamaarachchi)
III. Lab #2: Wet Lab Practice & Material Collection (April 15th)
- Micropipette Training; Experimental Workshop: Designing Controls (Collier / Walsh / Park)
- RNA Isolation, Centrifugal Spin-Based Isolation Techniques; Buffer Biochemistry (Collier / Walsh)
- DNA / RNA Quality: Fluorimetry & Spectrometry (Collier / Walsh)
IV. Introduction to Spatial Omics Methods (April 22nd)
- Spatial omics overview & multiplexed FISH [MERSCOPE, CosMx, Xenium] (Park)
- Antibody-based Spatial Proteomics Methods: CellScape, IMC, COMET, PhenoCycler, PixelGen (Park)
- Sequencing-based Spatial Omics Methods: Stereo-seq, VisiumHD, Slide-seq, Illumina spatial (Park)
- Reference atlas – Spatial Atlas of Human Anatomy (SAHA) project (Park)
V. Epigenetics & Epitranscriptomics (April 29th)
- Nanopore direct-RNA sequencing; -002 vs. -004 flow cells (Wilcox)
- Antibody-Based Epigenetics: ATAC-seq, HiC, CHIP-Seq, RAP-DNA (Mason)
- Class Project Introduction (Mason)
VI. Resources for Single-molecule Sequencing and Spatial Omics Analyses (May 6th)
- Machine Learning Assisted Methods for Biology (Hajirasouliha)
- Spatial multi-omics analysis and data integration (Segato Dezem)
- Downstream Analysis of direct-RNA seq: edgeR, DESeq2, PCA, Fisher’s Exact Test (Nelson)
VII. Lab #3: Preparing a Sequencing Library (May 13th)
- Experimental Workshop: Hypothesis Generation (Mason / Park)
- Single-molecule direct-RNA sequencing library prep; antibody-based capture (Collier / Walsh)
- Operation of sequencing technologies: Illumina, Nanopore, PacBio (Collier / Walsh)
VIII. Lab #4: Independent Nanopore direct-RNA Sequencing Analysis (May 20th)
- Bioconda, Package Installation, Data Management Practices (Nelson / Students)
- Basecalling: dorado; Alignment: minimap2; Processing: samtools and featureCounts (Students)
- Downstream Analysis of direct-RNA seq: DESeq2, PCA, Fisher’s Exact Test, eQTL-style analysis (Students)
IX. Single-Molecule Proteomic Sequencing & Genome Genetics-Ethics (May 27th)
- The Relationship Between Genetic Variation and Protein-Protein Interaction Networks (Michnick)
- New Spatial Omics Methods and Applications for Clinical and Biomedical Research (Rendeiro)
- The Ethics of Human and other Genome Engineering (Mason)
X. Lab #5: Refining & Interpreting Results (June 3rd)
- Mini-group sections with students & instructors to provide feedback.
XI. Student Research Presentation (June 10th)
- Students present results to instructors, subject specialists, and invited guests (oral presentation).