Single-Molecule & Spatial Sequencing: Methods & Applications

Overview

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.

Repository Structure

This repository is organized into course modules, each contained in separate folders corresponding to the course structure. Below is a description of each folder and its contents.

I. What is Single-Molecule Sequencing? (April 1st)

1. Sequencing technologies: Illumina, MGI, Sanger, Singular (no single molecules) – Mason
2. Course Overview & Sequencing technologies: Nanopore & PacBio; public data (single molecule) – Mason
3. 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)

4. Data wrangling and visualization in Python – Park / Nelson / Students
5. File Formats for Spatial and Single Cell Data: h5ad, RDS – Park
6. Single-molecule basecalling: pod5, slow5, blow5, SAM, BAM – Gamaarachchi

III. Lab #2: Wet Lab Practice & Material Collection (April 15th)

7. Micropipette Training; Experimental Workshop: Designing Controls – Collier / Walsh / Park
8. RNA Isolation, Centrifugal Spin-Based Isolation Techniques; Buffer Biochemistry – Collier / Walsh
9. DNA / RNA Quality: Fluorimetry & Spectrometry – Collier / Walsh

IV. Introduction to Spatial Omics Methods (April 22nd)

10. Spatial omics overview & multiplexed FISH (MERSCOPE, CosMx, Xenium) – Park
11. Antibody-based Spatial Proteomics Methods: CellScape, IMC, COMET, PhenoCycler, PixelGen – Park
12. Sequencing-based Spatial Omics Methods: Stereo-seq, VisiumHD, Slide-seq, Illumina spatial – Park
13. Reference atlas – Spatial Atlas of Human Anatomy (SAHA) project – Park

V. Epigenetics & Epitranscriptomics (April 29th)

14. Nanopore direct-RNA sequencing; -002 vs. -004 flow cells – Wilcox
15. Antibody-Based Epigenetics: ATAC-seq, HiC, CHIP-Seq, RAP-DNA – Mason
16. Class Project Introduction – Mason

VI. Resources for Single-molecule Sequencing and Spatial Omics Analyses (May 6th)

17. Machine Learning Assisted Methods for Biology – Hajirasouliha
18. Spatial multi-omics analysis and data integration – Segato Dezem
19. Downstream Analysis of direct-RNA seq: edgeR, DESeq2, PCA, Fisher’s Exact Test – Nelson

VII. Lab #3: Preparing a Sequencing Library (May 13th)

20. Experimental Workshop: Hypothesis Generation – Mason / Park
21. Single-molecule direct-RNA sequencing library prep; antibody-based capture – Collier / Walsh
22. Operation of sequencing technologies: Illumina, Nanopore, PacBio – Collier / Walsh

VIII. Lab #4: Independent Nanopore direct-RNA Sequencing Analysis (May 20th)

23. Bioconda, Package Installation, Data Management Practices – Nelson / Students
24. Basecalling: dorado; Alignment: minimap2; Processing: samtools and featureCounts – Students
25. 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)

26. The Relationship Between Genetic Variation and Protein-Protein Interaction Networks – Michnick
27. New Spatial Omics Methods and Applications for Clinical and Biomedical Research – Rendeiro
28. The Ethics of Human and other Genome Engineering – Mason

X. Lab #5: Refining & Interpreting Results (June 3rd)

29. Mini-group sections with students & instructors to provide feedback.

XI. Student Research Presentation (June 10th)

30. Students present results to instructors, subject specialists, and invited guests (oral presentation).

Getting Started

To get started with the course materials in this repository, clone the repository to your local machine using the following command:

git clone [https://github.com/Theo-Nelson/SMS-colab.git](https://github.com/Theo-Nelson/SMS-colab.git)
git