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Effect of Library Preparation on Differential Gene Expression in RNA Sequencing

Dang, Trang Le Thao (2023) Effect of Library Preparation on Differential Gene Expression in RNA Sequencing. Master's Thesis, University of Pittsburgh. (Unpublished)

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Library preparation can be a source of bias in RNA-sequencing studies, leading to false positives in genes identified as differentially expressed between conditions. This study aims to illustrate the impact of using different library preparations in RNA sequencing. Six adipose tissue RNA samples
from the Soifua Manuia project were sequenced in duplicate, with sequencing libraries prepared using both NEBNext Ultra RNA Library Prep Kit for Illumina (“standard preparation”) and
SMART-Seq v4 Ultra Low Input Kit for Sequencing for cDNA synthesis with Illumina Nextera XT library kit (“low-input preparation”). Differential gene expression analysis was performed with DESeq2, EdgeR, and Limma-Voom, and differentially expressed genes were annotated using BiomaRt.
Pathway analysis was performed with clusterProfiler, and visualization of all findings was generated using built-in functions of these tools or with ggplot2. All bioinformatics were conducted in R. There were 13827 differentially expressed genes, including ACTB and GADPH (housekeeping genes), identified in this study. Affected pathways included cellular respiration, regulation and localization of proteins, and ribosome biogenesis. These results indicate that library
preparation influences differential gene expression analysis and subsequently affects the downstream analyses that investigate the biological pathways impacted by gene expression level. These findings have public health relevance as transcriptomics studies become increasingly incorporated
into identifying factors for disease prevention and health promotion.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Dang, Trang Le Thaotld75@pitt.edutld75
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee MemberMinster, Ryan L.rminster@pitt.edurminster
Committee MemberFan,
Thesis AdvisorManna, Samanthaslr61@pitt.eduslr61
Date: 17 May 2023
Date Type: Publication
Defense Date: 24 April 2023
Approval Date: 17 May 2023
Submission Date: 28 April 2023
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 43
Institution: University of Pittsburgh
Schools and Programs: School of Public Health > Human Genetics
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: differential gene expression, RNA sequencing
Date Deposited: 17 May 2023 14:10
Last Modified: 17 May 2023 14:10


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