A Pipeline for Faecal Host DNA Analysis by Absolute Quantification of LINE-1 and Mitochondrial Genomic Elements Using ddPCR

Scientific Reports
Kuang HeMuneesh Tewari

Abstract

Stool contains DNA shed from cells of the gastrointestinal (GI) tract and has great potential as a bio-specimen for non-invasive, nucleic acid-based detection of GI diseases. Whereas methods for studying faecal microbiome DNA are plentiful, there is a lack of well-characterised procedures for stabilisation, isolation, and quantitative analysis of faecal host DNA. We report an optimised pipeline for faecal host DNA analysis from the point-of-collection to droplet digital PCR (ddPCR) absolute quantification of host-specific gene targets. We evaluated multiple methods for preservation and isolation of host DNA from stool to identify the highest performing methods. To quantify host DNA even if present in partially degraded form, we developed sensitive, human-specific short-amplicon ddPCR assays targeting repetitive nuclear genomic elements (LINE-1) and mitochondrial genes. We validated the ability of these optimised methods to perform absolute quantification of host DNA in 200 stool DNA extracts from samples that were serially collected from three healthy individuals and three hospitalised patients. These specimens allowed assessment of host DNA day-to-day variability in stool specimens with widely varying physical characteristics ...Continue Reading

References

Nov 5, 1997·Applied and Environmental Microbiology·I G Wilson
Mar 10, 2001·Nature·E S LanderUNKNOWN International Human Genome Sequencing Consortium
Jun 5, 2002·Genome Research·W James KentDavid Haussler
Aug 30, 2002·The International Journal of Biochemistry & Cell Biology·David G Nicholls
Jun 21, 2003·Clinical Chemistry·Kevin A BoyntonAnthony P Shuber
Feb 1, 1994·Trends in Cell Biology·M C PeitschJ Tschopp
Apr 30, 2005·Nature Reviews. Genetics·Robert W Taylor, Doug M Turnbull
Aug 18, 2005·Diagnostic Molecular Pathology : the American Journal of Surgical Pathology, Part B·Jeff OlsonAnthony P Shuber
Jun 16, 2006·Cancer Epidemiology, Biomarkers & Prevention : a Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology·Hongzhi ZouDavid A Ahlquist
Jun 16, 2012·Nature·UNKNOWN Human Microbiome Project Consortium
Jul 4, 2012·Journal of Applied Microbiology·C SchraderR Johne
Sep 14, 2012·Nature·Catherine A LozuponeRob Knight
Sep 3, 2013·Nature Methods·Christopher M HindsonMuneesh Tewari
Sep 5, 2013·Clinical Chemistry·Tanis C DingleKeith R Jerome
Feb 28, 2014·World Journal of Gastroenterology : WJG·Giulia De MaioDaniele Calistri
Apr 15, 2014·Expert Review of Molecular Diagnostics·Ruth Hall Sedlak, Keith R Jerome
Nov 14, 2014·Current Opinion in Gastroenterology·Andrew B ShreinerVincent B Young
Nov 25, 2014·Cell·Julia K GoodrichRuth E Ley
Dec 24, 2014·Nature Reviews. Genetics·Darren J Burgess
Jun 16, 2015·Cell Metabolism·Navdeep S Chandel
Jun 20, 2015·Journal of Immunology Research·Caroline VincentAmee R Manges
Jul 15, 2015·The Science of the Total Environment·Xiwei HeXu-Xiang Zhang
Oct 27, 2015·The FEBS Journal·Maria Eugenia DelgadoThomas Brunner
Jan 15, 2016·Cell Host & Microbe·Shirong LiuHoward L Weiner
Feb 3, 2016·Cold Spring Harbor Perspectives in Biology·Huda Y Zoghbi, Arthur L Beaudet
Feb 24, 2016·ELife·Ed ReznikChris Sander
Aug 22, 2017·Nature Reviews. Genetics·Andrew Brantley HallRamnik J Xavier
Nov 29, 2017·Tumour Biology : the Journal of the International Society for Oncodevelopmental Biology and Medicine·Bhupender KumarM Moshahid Alam Rizvi
Jun 15, 2018·BMJ : British Medical Journal·Ana M ValdesTim D Spector
Nov 6, 2018·BioTechniques·Mark F Kavlick

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Citations

Nov 20, 2020·International Journal of Molecular Sciences·Zuzana PösTomas Szemes
Sep 3, 2021·Bioelectrochemistry·Samaneh Hadian-GhazviniHedayatolah Ghourchian

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Methods Mentioned

BETA
PCR
xenograft
electrophoresis
Assay

Software Mentioned

TapeStation
QuantaSoft

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