Annotation and Expression Analysis of HERV Proviruses
HERV proviruses and other repeat regions were annotated as previously described58 (link). In brief, hidden Markov models (HMMs) representing known human repeat families (Dfam 2.0 library v.150923) were used to annotate GRCh38 using RepeatMasker, configured with nhmmer. RepeatMasker annotates long terminal repeats (LTRs) and internal regions separately; thus, tabular outputs were parsed to merge adjacent annotations for the same element. A list of HERV proviruses with functional env ORFs was compiled (Supplementary Table 1), and RNA-seq reads from TCGA, GTEx and TRACERx were mapped and counted using a custom transcriptome assembled on a subset of the RNA-seq data from TCGA, as previously described58 (link). In brief, TPM values were calculated for all transcripts in the transcript assembly with a custom Bash pipeline using GNU parallel and Salmon (v.0.12.0)59 (link). TPM values were then imported into Qlucore Omics Explorer v.3.3 (Qlucore) for downstream differential expression analysis and visualization. In the case of multiple transcripts transcribed from a given HERV provirus, data were collapsed by summing expression of any of the multiple transcripts overlapping the env ORF of that provirus. Patient-level mean values were calculated across multiple primary tumour regions, as applicable.
Ng K.W., Boumelha J., Enfield K.S., Almagro J., Cha H., Pich O., Karasaki T., Moore D.A., Salgado R., Sivakumar M., Young G., Molina-Arcas M., de Carné Trécesson S., Anastasiou P., Fendler A., Au L., Shepherd S.T., Martínez-Ruiz C., Puttick C., Black J.R., Watkins T.B., Kim H., Shim S., Faulkner N., Attig J., Veeriah S., Magno N., Ward S., Frankell A.M., Al Bakir M., Lim E.L., Hill M.S., Wilson G.A., Cook D.E., Birkbak N.J., Behrens A., Yousaf N., Popat S., Hackshaw A., Hiley C.T., Litchfield K., McGranahan N., Jamal-Hanjani M., Larkin J., Lee S.H., Turajlic S., Swanton C., Downward J, & Kassiotis G. (2023). Antibodies against endogenous retroviruses promote lung cancer immunotherapy. Nature, 616(7957), 563-573.
Herv Human Library Long terminal repeats Multiple primary tumour Orfs Patient Provirus Rna seqSalmonTranscriptome
Corresponding Organization :
Other organizations :
The Francis Crick Institute, Samsung Medical Center, Sungkyunkwan University, Cancer Research UK, London Cancer, CRUK Lung Cancer Centre of Excellence, University College London, Samsung (South Korea), Royal Marsden Hospital, Royal London Hospital, Imperial College London, Genomics (United Kingdom), University College Hospital, The Patients Association, Institute of Structural and Molecular Biology, Harefield Hospital, Guy's and St Thomas' NHS Foundation Trust, Royal Free London NHS Foundation Trust, Institute of Cancer Research, Whittington Hospital, Aberdeen Royal Infirmary, NHS Grampian, University of Birmingham, University of Leicester, Cancer Research UK Scotland Institute, University of Manchester, University Hospital Southampton NHS Foundation Trust, University of Aberdeen, The Christie NHS Foundation Trust, Golden Jubilee National Hospital, Liverpool Heart and Chest Hospital, University of Liverpool, NHS Greater Glasgow and Clyde, Princess Alexandra Hospital NHS Trust, Royal Surrey NHS Foundation Trust, Sheffield Teaching Hospitals NHS Foundation Trust, Swansea Bay University Health Board, Singleton Hospital, University Hospitals Birmingham NHS Foundation Trust, University Hospitals of Leicester NHS Trust, Manchester University NHS Foundation Trust, Wythenshawe Hospital, Harvard University, Mass General Brigham, Max Delbrück Center, Aarhus University, Boston Children's Hospital, Oncode Institute, The Netherlands Cancer Institute, The University of Texas MD Anderson Cancer Center, Royal Marsden NHS Foundation Trust
Hidden Markov models (HMMs) representing known human repeat families (Dfam 2.0 library v.150923)
RepeatMasker configuration with nhmmer
dependent variables
Annotation of HERV proviruses and other repeat regions
Identification of HERV proviruses with functional env ORFs
Expression levels (TPM values) of HERV provirus transcripts
control variables
GRCh38 genome assembly
Subset of RNA-seq data from TCGA used for custom transcriptome assembly
positive controls
None specified
negative controls
None specified
Annotations
Based on most similar protocols
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