HSLS MolBio Workshops

This workshop briefly introduces techniques, platforms, and methods used in bulk RNA-Seq experiments, followed by a software demonstration using the HSLS-licensed CLC Genomics Workbench.

Participants will learn how to

• access the CLCbio Genomics Server hosted on the HTC Cluster by Pitt CRC
• import RNA-Seq FASTQ reads from a GEO dataset
• assess the quality of RNA-Seq data
• align reads to a reference genome
• estimate known gene and transcript expression
• perform differential expression analysis
• visualize data by generating PCA and heatmaps

Target Audience

Experimental biologists seeking to analyze bulk RNA-Seq data generated through experiments or retrieved from a repository such as GEO. The software covered in the workshop operates through a user-friendly, point-and-click graphical user interface, so neither programming experience nor familiarity with the command line interface is required.

Powerpoints

• RNA-Seq w/ CLC-Gx (ppt) - main presentation slides
• Practice Exercise - A set of practice exercise problems on bulk RNA-seq data analysis

Lecture Videos

• BulkRNA-seq Data Analysis using CLC Genomics Workbench

Data Download

• ENA Browser for SRP113442 - link to download FASTQ reads for AI vs DMSO dataset
• Metadata file (.xlsx) - link to download a metadata file for AI vs DMSO dataset
• Practice exercise dataset: Human Airway Smooth Muscle Transcriptome Changes in Response to Asthma Medications (GSE52778) 
• Metadata file  for the practice exercise - link to download a metadata file for Dex vs Unt dataset (GSE52778)
• Hands-on exercise (DEX vs. Unt) CLCGW project: A folder with sequence reads, mapping results, and DEG data to import 
• Human hg38 reference Sequence

Software Registration

• CLC Genomics workbench - to receive the software download link and licensing information

• CRC-CLCBio Genomics server account setup - Instructions to connect to the CLCBio Genomics Server hosted at the Center for Research Computing (CRC)

Video Tutorials

• How to import reads (FASTQ files) into CLC
• How to create a Metadata Table
• How to create an Expression Browser
• FPKM, RPKM, and TPM explained (StatQuest)
• Hierarchical Clustering explained (StatQuest)
• PCA Explained in 5 min (StatQuest)
• False Discovery Rate (FDR) clearly explained (StatQuest) 

Documentations

• CLC Genomics workbench v24.0.2 Manual 
• Statistics for Biologists -a collection highlights important statistical issues that biologists should be aware of and provides practical advice to help them improve the rigor of their work.
• FastQC Problems and Discussion 
• RNA Seqlopedia - provides an overview of RNA-seq and of the choices necessary to carry out a successful RNA-seq experiment.
• RNA-seq data analysis using R (HavardX) - MOOC for biomedical data science
• RNAbio - RNASeq data analysis basics
• Data transfer using Ondemand tool in CRC

• SequencEnG, an interactive learning resource for NGS.
• ENA (Europian Nucleotide Archive)
• Ensembl Reference Sequences
• FASTQC - a quality control tool for high throughput sequence data
• NCBI GEO - RNA-seq dataset from  Series GSE101788
• NCBI SRA (Sequence Read Archive) 
• Venn Diagram Creator 
• BioRender - Scientific illustrations creating tool

Featured Article

• Martin, B., Gabris-Weber, B. A., Reddy, R., Romero, G., Chattopadhyay, A. and Salama, G. (2018). Relaxin reverses inflammatory and immune signals in aged hearts. Plos One 13(1), e0190935. 

Key References

• Pulikkan JA, Hegde M, Ahmad HM, et al. CBFβ-SMMHC Inhibition Triggers Apoptosis by Disrupting MYC Chromatin Dynamics in Acute Myeloid Leukemia. Cell. 2018;174(1):172-186.e21. doi:10.1016/j.cell.2018.05.048 (dataset used for the workshop)
• Stark, R., Grzelak, M. & Hadfield, J. RNA sequencing: the teenage years. Nat. Rev. Genet. (2019). doi:10.1038/s41576-019-0150-2
• Chatterjee A, Ahn A, Rodger EJ, Stockwell PA, Eccles MR. A Guide for Designing and Analyzing RNA-Seq Data. Methods Mol Biol. 2018;1783:35-80. doi:10.1007/978-1-4939-7834-2_3
• Conesa, A. et al. A survey of best practices for RNA-seq data analysis. Genome Biol. 17, 13 (2016).
• Benchmark document on the the performance of the Differential Expression Analysis tool in comparison to well-accepted protocols like DEseq, EdgeR, etc.
• Simoneau, J., Dumontier, S., Gosselin, R. & Scott, M. S. Current RNA-seq methodology reporting limits reproducibility. Brief. Bioinformatics (2019). doi:10.1093/bib/bbz124 

Publications citing CLC-Genomics Workbench from Pitt researchers (346 as of January 17, 2024)

Attribution

Please include the following statement in the acknowledgments section for all publications, posters, and presentations: 

CLC Genomics Workbench software licensed through the Molecular Biology Information Service of the Health Sciences Library System, University of Pittsburgh  (RRID:SCR_011975) was used for data analysis.

If you used the Pitt-CRC server for CLC Genomics Workbench, then please also include the following text in the acknowledgments section for all publications, posters, and presentations: 

“This research was supported in part by the University of Pittsburgh Center for Research Computing​, RRID:SCR_022735, through the resources provided. Specifically, this work used the HTC cluster, which is supported by NIH award number S10OD028483."

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