TY - BOOK
T1 - High throughput sequencing in human cancer tissue
T2 - viral metagenomics & miRNA profiling
AU - Asplund, Maria
PY - 2017
Y1 - 2017
N2 - High throughput sequencing (HTS) is a sequence independent and sensitive method applicable ina wide range of genomic research. Since its introduction in the beginning of the 21st century ithas greatly accelerated the amount of sequencing data produced. In life sciences, includingcancer research, HTS has opened up for new kinds of studies including large sample sets andgenome and transcriptome wide investigations. The aim of this thesis was to investigate theapplicability and challenges of using HTS for genomic investigations in human (cancer) tissuesamples. This was done using two separate design strategies: 1) viral metagenomics in humancancer tissue samples and selected control samples, and 2) miRNA profiling in basal cellcarcinoma (BCC) samples and healthy control samples. In the first study from the viralmetagenomic strategy, more than 15 cancer types were screened for viruses using HTS. Withnine different viral enrichment methods this is the first study targeting all major viral classes,including both DNA and RNA viruses, in multiple cancer types. Generating more than 700sequencing libraries the viromes of nearly 200 cancer tissue samples were investigated. Virusesidentified included predominantly papillomaviruses, anelloviruses, herpesviruses, andparvoviruses. Critical awareness points in viral metagenomic studies, such as cross-speciesmapping, bleed-over, and viral artifacts were also presented. In the second study from the viralmetagenomic strategy, cancer samples, virus spiked samples and non-template control sampleswere investigated for method related viral contamination. This resulted in a catalogue of morethan 500 contaminating viral sequences linked to laboratory components. We thereby showedthat reagents and utilities used for enrichment of viral sequences and library preparation containa diversity of viral sequences, not before presented. The viral contaminating sequences weremostly of non-human viral origin but contaminating human viral sequences were also identified.In the study from the miRNA profiling strategy, HTS was applied to obtain transcriptome widemiRNA profiles from 22 BCC samples and 22 paired healthy control samples from the samepatients. Read sequences were mapped to split pre-miRNA hairpin sequences, identifying morethan 100 differentially expressed miRNAs not previously presented in relation to BCC. Resultswere validated by comparison to parallel results from mapping to mature miRNA sequences.Altogether, this thesis has shown the strength of HTS as a sensitive method generatingcomprehensive results. Moreover, It has highlighted some critical awareness points such ascontamination and the importance of bioinformatic confirmation using parallel/complementaryanalysis methods.
AB - High throughput sequencing (HTS) is a sequence independent and sensitive method applicable ina wide range of genomic research. Since its introduction in the beginning of the 21st century ithas greatly accelerated the amount of sequencing data produced. In life sciences, includingcancer research, HTS has opened up for new kinds of studies including large sample sets andgenome and transcriptome wide investigations. The aim of this thesis was to investigate theapplicability and challenges of using HTS for genomic investigations in human (cancer) tissuesamples. This was done using two separate design strategies: 1) viral metagenomics in humancancer tissue samples and selected control samples, and 2) miRNA profiling in basal cellcarcinoma (BCC) samples and healthy control samples. In the first study from the viralmetagenomic strategy, more than 15 cancer types were screened for viruses using HTS. Withnine different viral enrichment methods this is the first study targeting all major viral classes,including both DNA and RNA viruses, in multiple cancer types. Generating more than 700sequencing libraries the viromes of nearly 200 cancer tissue samples were investigated. Virusesidentified included predominantly papillomaviruses, anelloviruses, herpesviruses, andparvoviruses. Critical awareness points in viral metagenomic studies, such as cross-speciesmapping, bleed-over, and viral artifacts were also presented. In the second study from the viralmetagenomic strategy, cancer samples, virus spiked samples and non-template control sampleswere investigated for method related viral contamination. This resulted in a catalogue of morethan 500 contaminating viral sequences linked to laboratory components. We thereby showedthat reagents and utilities used for enrichment of viral sequences and library preparation containa diversity of viral sequences, not before presented. The viral contaminating sequences weremostly of non-human viral origin but contaminating human viral sequences were also identified.In the study from the miRNA profiling strategy, HTS was applied to obtain transcriptome widemiRNA profiles from 22 BCC samples and 22 paired healthy control samples from the samepatients. Read sequences were mapped to split pre-miRNA hairpin sequences, identifying morethan 100 differentially expressed miRNAs not previously presented in relation to BCC. Resultswere validated by comparison to parallel results from mapping to mature miRNA sequences.Altogether, this thesis has shown the strength of HTS as a sensitive method generatingcomprehensive results. Moreover, It has highlighted some critical awareness points such ascontamination and the importance of bioinformatic confirmation using parallel/complementaryanalysis methods.
UR - https://rex.kb.dk/primo-explore/fulldisplay?docid=KGL01011006398&context=L&vid=NUI&search_scope=KGL&tab=default_tab&lang=da_DK
M3 - Ph.D. thesis
BT - High throughput sequencing in human cancer tissue
PB - Natural History Museum of Denmark, Faculty of Science, University of Copenhagen
ER -
