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 SCHEDULE
I. What is Single-Molecule Sequencing? (April 2nd)
1. Sequencing technologies: Illumina, MGI, Sanger, Singular [no single molecules] (Mason)
2. Course Overview & Sequencing technologies: Nanopore & PacBio; public data [single molecule] (Mason)
3. 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)
22. Systems Biology Approaches to Transforming Human Health and Longevity (Snyder)
23. Single-molecule proteomic sequencing: data formats, wet-lab methods, and dry-lab analysis (TBD)
24. 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.