COURSE DESCRIPTION
Sequencing-based research has become the dominant investigative practice within the biological sciences. Single-molecule sequencing: training, methods, and 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 TECHNOLOGY
Throughout this course you will learn to use various tools and databases including BLAST, 
, Shiny Server, One Codex, and many more!
To access assignments, students are encouraged to register for a Google Account (https://www.google.com/) and ChatGPT account (https://chat.openai.com/), or employ their preferred generative AI service.
COURSE SCHEDULE
I. What is Single-Molecule Sequencing?
April 2nd
- Sequencing technologies: Illumina, MGI, Sanger, Singular [not single molecule] (Mason)
- Course Overview & Sequencing technologies: Nanopore & PacBio; public data [single molecule] (Mason)
- Experimental Design & Single-Molecule Fluorescence Sequencing (Nelson)
II. Lab #1: Dry Lab Practice & Single-Molecule Sequencing Formats
April 9th
- 4. Terminal, Command Line, Linux, ChatGPT, TSV, CSV, FASTQ file formats (Nelson)
- 5. Single-molecule DNA & RNA base calling: fast5, slow5, blow5, dorado, SAM, BAM (Nelson)
- 6. Data wrangling and visualization in R: vcf, tidyverse, ggplot2 (Sienkiewicz)
III. Epigenomes, DNA Modifications, and Single-Molecule DNA-sequencing
April 16th
- 7. Single-molecule DNA-base calling accuracy vs. variability; sources of signal noise (Mason)
- 8. Introduction to DNA-sequencing source variants: ATAC-seq, HiC, CHIP-Seq, RAP-DNA (Mason)
- 9. Next-gen sequencing analysis: metagenomic sequencing analysis workshop (Tierney)
IV. Lab #2: Wet Lab Practice & Material Collection
April 23rd
- 10. Micropipette Training; Experimental Workshop: Designing Controls (Nelson / Ryon)
- 11. RNA Isolation, Centrifugal Spin-Based Isolation Techniques; Buffer Biochemistry (Nelson / Ryon)
- 12. DNA / RNA Quality: Fluorimetry & Spectrometry (Nelson / Ryon)
V. Epitranscriptomics, RNA Modifications, and Single-Molecule RNA-sequencing
April 30th
- 13. Nanopore direct-RNA sequencing; -002 vs. -004 flow cells (Mason)
- 14. Downstream Analysis of direct-RNA seq: edgeR, DESeq2, PCA, Fisher’s Exact Test (Nelson)
- 15. Class Project Introduction (Mason)
VI. Lab #3: Preparing a Sequencing Library
May 7th
- 16. Experimental Workshop: Hypothesis Generation (Mason)
- 17. Single-molecule direct-RNA sequencing library prep; antibody-based capture (Nelson / Ryon)
- 18. Operation of sequencing technologies: Illumina, Nanopore, PacBio, QuantumSI (Ryon)
VII. Machine Learning and Systems Biology
May 14th
- 19. Spatial Omics Methods and Applications (Schwartz)
- 20. Class Project Recap & Poster Presentation Tips (Mason)
- 21. Multi-omic sequencing data integration: batch effects, challenges, unsupervised learning (Sienkiewicz)
VIII. Lab #4: Independent Nanopore direct-RNA sequencing Analysis
May 21st
- 22. Bioconda, Package Installation, Wasabi, Cloud Data Transfers; wasabi (Students)
- 23. Basecalling: dorado; Alignment: minimap2; Processing: samtools and featureCounts (Students)
- 24. Downstream Analysis of direct-RNA seq: edgeR, DESeq2, PCA, Fisher’s Exact Test, eQTL-style analysis (Students)
IX. Single-Molecule Proteomic Sequencing & Genome Genetics-Ethics
May 28th
- 25. Systems Biology Approaches to Transforming Human Health and Longevity (Snyder)
- 26. Single-molecule proteomic sequencing: data formats, wet-lab methods, and dry-lab analysis (TBD)
- 27. The Ethics of Human and other Genome Engineering (Mason)
X. Student Poster Presentation
June 4th
- 25. Students present posters to instructors, subject specialists, and invited guests.