Beruflich Dokumente
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Introduction
Results
50 bp
ladder
Targe)ng
the
shorter
V6
region
of
the
16S
rRNA
gene
gives
closer
results
between
FFPE
and
frozen
samples
than
when
targe)ng
the
V4
region.
Phylum
Phylum
Genera
Genera
Samples
1-61-6
V4
FFPE samples
V4
Samples 1-6
Samples
1-61-6
V6
FFPE samples
Negative
Negative
controls
controls
:Nc1 (Agar), N7 Taxonomy summary
(3/20
(95% ethanol)
and
produced
N14 (tap
water).
50 bp
ladder
50 bp
ladder
Samples
1-6 1-6
V4
frozen samples
Aim
50 bp
ladder
V6 Negative
controls (4/20
Negative controls
:Nc1
produced
(Agar), N2 (formalin)
libraries)
and N9 (ethanol)
and
N12 (xylene).
libraries)
Phylum
Figure
2.
Frozen
and
FFPE
taxonomy
bar
graph.
Graph
showing
the
rela^ve
abundances
of
bacteria
at
genera
level.
This
shows
that
FFPE
and
frozen
results
for
each
individual
correlate.
However,
a
signicant
dierence
in
beta
diversity
between
FFPE
and
frozen
samples
was
found.
Results
correlated
beTer
between
FFPE
and
frozen
when
targe^ng
V6
compared
to
V4.
Seven
out
of
forty
nega^ve
controls
were
contaminated.
These
are
dis^nguishable
on
this
graph
from
each
other
and
the
faecal
samples.
Genera
Data
from
frozen
samples
varied
signicantly
based
on
the
varible
region
targeted.
Longer
regions
tended
to
detect
a
higher
level
of
alpha
diversity.
PC1 7%
Methods
Taxonomy summary
Phylum
V6
V7/V8
Legend
Genera
V2
V3
V4
V5
V6
V7/V8
Figure 1. Taxonomy area graphs. Area graphs showing the relative abundances of bacteria at
phylum level and genera level. Legend contains all phylum shown to vary significantly based on
variable region targeted. Many species in the legend are not
Legend
Figure
4.
Taxonomy
area
graphs.
Area
graphs
showing
the
rela^ve
abundances
of
bacteria
at
phylum
and
genera
level
according
to
variable
region
of
16S
rRNA
gene
targeted.
The
listed
phyla
varied
in
abundance
signicantly
dependent
on
region
targeted
(p
=
<
0.002).
Figure 1. Taxonomy area graphs. Area graphs showing the relative abundances of bacteria at
phylum level and genera level. Legend contains all phylum shown to vary significantly based on
variable region targeted. Many species in the legend are not
Conclusions
References
1.
2.
3.
www.virtualpathology.leeds.ac.uk
VirtualPathology@leeds.ac.uk
This
is
the
rst
study
to
apply
NGS
technology
to
FFPE
faecal
material.
We
have
successfully
produced
microbiome
data
from
FFPE
material
comparable
to
that
from
frozen
samples.
When
targe)ng
the
V6
region
there
is
a
closer
correla)on
between
frozen
and
FFPE
samples
than
when
targe)ng
V4.
We
hypothesise
that
this
is
due
to
the
shorter
V6
region
being
less
aected
by
DNA
fragmenta)on.
Microbiome
analysis
in
frozen
material
is
signicantly
aected
by
the
variable
region
of
the
16s
rRNA
gene
targeted.
It
is
clear
that
choice
of
material
and
variable
region
is
important
in
microbiome
studies
and
further
research
is
needed
to
standardise
best
prac)se
when
studying
the
gut
microbiome.
Section of Pathology and Tumour Biology, Leeds Institute of Cancer and Pathology,
University of Leeds
www.youtube.com/
LeedsPathology
@LeedsPathology