Dolphin
Senior Member (Voting Rights)
Only a small part of this appears to be on ME/CFS
From: Dr. Marc-Alexander Fluks
Source: University of East Anglia
Date: December 2020
URL: https://ueaeprints.uea.ac.uk/id/eprint/81750/
https://ueaeprints.uea.ac.uk/id/eprint/81750/1/2020HsiehS-YPhD.pdf
Investigating the human intestinal virome
-----------------------------------------
Shen-Yuan Hsieh
- Quadram Institute Biosciences, Faculty of Medicine and Health
Sciences, Norwich Medical School. University of East Anglia, U.K.
Abstract
The human intestinal virobiota consists of highly complex and diverse
viruses and virus-associated genes (termed the 'virome'), dominated by
bacteriophages. Numerically, viruses have been considered the most
abundant and diverse biological entities on Earth, estimated to be
approximately 1031 in number. In the human gastrointestinal tract (GIT),
virus-to-microbe ratio (VMR) may be close to 1:1, while it may reach
20:1 at mucosal surfaces and within the mucus layer, in total numbering
1010-1015 virus-like particles (VLPs). Recent studies suggested that
changes in the intestinal virome may lead to chronic GI-inflammation and
intestinal microbial dysbiosis, thereby triggering diseases such as
myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Thus, this
thesis aimed to develop robust and reliable protocols for characterising
the human faecal microbiome that can be applied to analysing the virome
in patients with severe ME/CFS.
This thesis first aimed to develop a reliable and reproducible protocol
for VLP isolation from human faeces and for VLP quantification using a
digital image analysis (DIA)-based method. Protocols were then optimised
for VLP DNA extraction to obtain DNA of sufficient quality and quantity
for next generation sequencing (NGS). As part of these studies, I
developed a bioinformatics pipeline for viral metagenomic analysis, and
also, I determined the extent of PCR amplification bias in
virome-enriched, uncultivated virus genomes (UViGs) by comparing the
methods of linker amplified shotgun library (LASL) and non-amplified
shotgun library (NASL) preparations. The optimised protocols and
bioinformatics pipeline were then applied to the initial analysis of the
faecal virome of severely affected ME/CFS patients and same household
healthy control individuals (SHHC).
The optimised protocol is comprised of (1) homogenisation of faecal
samples by vortexing without the use of bead-beating, followed by
incubation on ice to facilitate the release of VLPs from solid
materials; (2) partition of crude faecal matter, dietary debris and
virions/VLPs by two-round of high-speed centrifugation; (3) sequential
filtration using 0.8 mum and 0.45 mum filter; (4) PEG precipitation; (5)
DNase and RNase treatment; (6) proteinase K digestion; (7) viral capsid
lysis with SDS lysis buffer; (8) Phenol/Chloroform/Isoamyl alcohol
extraction; (9) DNA purification using silica-based spin columns, and
(10) DNA concentration using a vacuum-based condenser. Using three
independent stool samples to evaluate reproducibility, VLP DNA yields
were between 67.2 ng and 94.8 ng per gram of faeces. For VLP
quantification, manual counting-based DIA method was more accurate and
reliable than automated counting-based method.
Using an optimised bioinformatics pipeline to analyse UViGs from PCR and
non-PCR virome-derived datasets, I found that misrepresentation of
certain viruses may occur after amplification in their relative
abundance. In alpha diversity, the UViGs from non-PCR datasets generally
have higher richness and diversity than those from PCR datasets,
suggesting that PCR is likely to lower viral richness and diversity.
Moreover, the major differences in beta diversity were more likely to be
driven by a high level of intestinal virome individuality between
donors, while amplification bias may have a minor effect on the beta
diversity of viruses in the PCR datasets. In addition, in an initial
analysis of comparing UViG similarity networks, I found that there is no
significant difference between both datasets but further investigation
is required.
In comparing faecal samples from ME/CFS and SHHC, VLPs with nucleic
acid-containing capsids in SHHC samples were higher than those of severe
ME/CFS patients, although the variation and diversity of VLP were seen
in both sets of faecal samples. Transmission electron microscopy (TEM)
analysis identified Siphoviridae as the most prominent virus in both
ME/CFS patients and SHHC VLP samples. Moreover, giant Siphoviruses were
occasionally detected, suggesting potential novel strains are present in
these samples. The biological meaning of these findings is not clear and
requires further investigation. In ongoing work, the optimised protocol
and bioinformatic pipeline is being applied to investigating the
composition of the intestinal virome in severe ME/CFS patients and SHHC.
--------
(c) 2020 University of East Anglia
From: Dr. Marc-Alexander Fluks
Source: University of East Anglia
Date: December 2020
URL: https://ueaeprints.uea.ac.uk/id/eprint/81750/
https://ueaeprints.uea.ac.uk/id/eprint/81750/1/2020HsiehS-YPhD.pdf
Investigating the human intestinal virome
-----------------------------------------
Shen-Yuan Hsieh
- Quadram Institute Biosciences, Faculty of Medicine and Health
Sciences, Norwich Medical School. University of East Anglia, U.K.
Abstract
The human intestinal virobiota consists of highly complex and diverse
viruses and virus-associated genes (termed the 'virome'), dominated by
bacteriophages. Numerically, viruses have been considered the most
abundant and diverse biological entities on Earth, estimated to be
approximately 1031 in number. In the human gastrointestinal tract (GIT),
virus-to-microbe ratio (VMR) may be close to 1:1, while it may reach
20:1 at mucosal surfaces and within the mucus layer, in total numbering
1010-1015 virus-like particles (VLPs). Recent studies suggested that
changes in the intestinal virome may lead to chronic GI-inflammation and
intestinal microbial dysbiosis, thereby triggering diseases such as
myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Thus, this
thesis aimed to develop robust and reliable protocols for characterising
the human faecal microbiome that can be applied to analysing the virome
in patients with severe ME/CFS.
This thesis first aimed to develop a reliable and reproducible protocol
for VLP isolation from human faeces and for VLP quantification using a
digital image analysis (DIA)-based method. Protocols were then optimised
for VLP DNA extraction to obtain DNA of sufficient quality and quantity
for next generation sequencing (NGS). As part of these studies, I
developed a bioinformatics pipeline for viral metagenomic analysis, and
also, I determined the extent of PCR amplification bias in
virome-enriched, uncultivated virus genomes (UViGs) by comparing the
methods of linker amplified shotgun library (LASL) and non-amplified
shotgun library (NASL) preparations. The optimised protocols and
bioinformatics pipeline were then applied to the initial analysis of the
faecal virome of severely affected ME/CFS patients and same household
healthy control individuals (SHHC).
The optimised protocol is comprised of (1) homogenisation of faecal
samples by vortexing without the use of bead-beating, followed by
incubation on ice to facilitate the release of VLPs from solid
materials; (2) partition of crude faecal matter, dietary debris and
virions/VLPs by two-round of high-speed centrifugation; (3) sequential
filtration using 0.8 mum and 0.45 mum filter; (4) PEG precipitation; (5)
DNase and RNase treatment; (6) proteinase K digestion; (7) viral capsid
lysis with SDS lysis buffer; (8) Phenol/Chloroform/Isoamyl alcohol
extraction; (9) DNA purification using silica-based spin columns, and
(10) DNA concentration using a vacuum-based condenser. Using three
independent stool samples to evaluate reproducibility, VLP DNA yields
were between 67.2 ng and 94.8 ng per gram of faeces. For VLP
quantification, manual counting-based DIA method was more accurate and
reliable than automated counting-based method.
Using an optimised bioinformatics pipeline to analyse UViGs from PCR and
non-PCR virome-derived datasets, I found that misrepresentation of
certain viruses may occur after amplification in their relative
abundance. In alpha diversity, the UViGs from non-PCR datasets generally
have higher richness and diversity than those from PCR datasets,
suggesting that PCR is likely to lower viral richness and diversity.
Moreover, the major differences in beta diversity were more likely to be
driven by a high level of intestinal virome individuality between
donors, while amplification bias may have a minor effect on the beta
diversity of viruses in the PCR datasets. In addition, in an initial
analysis of comparing UViG similarity networks, I found that there is no
significant difference between both datasets but further investigation
is required.
In comparing faecal samples from ME/CFS and SHHC, VLPs with nucleic
acid-containing capsids in SHHC samples were higher than those of severe
ME/CFS patients, although the variation and diversity of VLP were seen
in both sets of faecal samples. Transmission electron microscopy (TEM)
analysis identified Siphoviridae as the most prominent virus in both
ME/CFS patients and SHHC VLP samples. Moreover, giant Siphoviruses were
occasionally detected, suggesting potential novel strains are present in
these samples. The biological meaning of these findings is not clear and
requires further investigation. In ongoing work, the optimised protocol
and bioinformatic pipeline is being applied to investigating the
composition of the intestinal virome in severe ME/CFS patients and SHHC.
--------
(c) 2020 University of East Anglia
