Investigating the faecal microbiome in

formalin fixed paraffin embedded (FFPE)

material
Ivan Jobling, Morag Taylor, Caroline Young, Henry Wood, Phil Quirke
Section of Pathology & Tumour Biology, Leeds Institute of Cancer and Pathology, University of Leeds

Introduction

Results

The microbiome describes the

e c o l o g i c a l c o m m u n i t y o f
c o m m e n s a l , s y m b i o ^ c a n d
pathogenic microorganisms that
share our body space (1).
Altered faecal microbiomes are
present in a range of gastrointes^nal
pathologies but their exact role in
disease is not yet known (2,3).
C u r r e n t l y r e s e a r c h i n t o t h e
microbiome makes use of fresh or
frozen samples.
One poten^al method for rapidly
expanding and diversifying research
is the retrospec^ve study of formalin
xed paran embedded (FFPE)
samples that contain faecal bacterial
popula^ons.

It is possible to successfully amplify

the 16S rRNA gene from FFPE
faecal material.

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

V6 Frozen 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).

Phylum with abundance over 1%

To inves)gate the feasibility of typing

the microbiome in FFPE faecal samples
and analyse how microbiome analysis
varied when targe)ng dierent
variable regions.

Figure 1. Gel electrophoresis. Gel showing

amplica^on of the V6 region of the 16S rRNA
gene in FFPE and frozen faecal samples.
Reference ladders contain DNA strands 50 base
pairs (bp) apart in size. Nega^ve and posi^ve
controls shown. Figure shows that it is possible
to successfully amplify the 16S rRNA gene from
FFPE faecal material.

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).

Figure 3. Alpha diversity. Rarefac^on chart showing

the dierence in within sample diversity between
FFPE and frozen samples against sequences per
sample. There is a signicant dierence between V4
FFPE and frozen samples (p = 0.05) but not V6.

libraries)

Sample source (faecal or negative control)

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

FFPE blocks were created from six

anonymised frozen faecal samples.
The V4 (240 base pair product) and V6 (98
base pair product) regions of the 16S rRNA
gene were amplied in FFPE samples.
For frozen samples (including from 57 further
samples) regions V2, V3, V5, V7/V8 were also
amplied.

Phylum

V6

V7/V8

Legend

Genera

Libraries were prepared from PCR product

and samples submiTed for NGS using an
Illumina MiSeq.
QIIME soZware was used for analysis. The
dierent bacterial phylum and genera
present were collated for each sample group.
Alpha (within sample) and beta (between
sample) diversity were calculated. s

Phyla listed had signicantly

dierent abundance dependent
on variable region
of
RNA V5
V2
V316S rV4
gene targeted.

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 5. Alpha diversity of frozen samples

according to variable region targeted. Graph shows
signicant dierences between all variable regions
targeted with shorter regions detec^ng less
diversity (p < 0.001).

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.

Lederberg J, McCray AT. 'Ome sweet 'omics - A genealogical treasury

of words. Scientist. 2001 Apr 2;15(7):8. PubMed PMID: WOS:
000168167400002.

2.

Greenblum S, Turnbaugh PJ, Borenstein E. Metagenomic systems

biology of the human gut microbiome reveals topological shifts
associated with obesity and inflammatory bowel disease. Proceedings
of the National Academy of Sciences of the United States of America.
2012 Jan 10;109(2):594-9. PubMed PMID: 22184244. Pubmed
Central PMCID: 3258644.

3.

Chang JY, Antonopoulos DA, Kalra A, Tonelli A, Khalife WT, Schmidt

TM, et al. Decreased diversity of the feacal microbiome in recurrent
Clostridium difficile-associated diarrhea. The Journal of infectious
diseases. 2008 Feb 1;197(3):435-8. PubMed PMID: 18199029.

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