Technical challenges regarding the use of formalin-fixed paraffin embedded (FFPE) tissue specimens for the detection of bacterial alterations in colorectal cancer.
Colorectal neoplasms
DNA contamination
Formalin-fixed paraffin embedded
Gastrointestinal microbiome
High-throughput nucleotide sequencing
Low biomass
Journal
BMC microbiology
ISSN: 1471-2180
Titre abrégé: BMC Microbiol
Pays: England
ID NLM: 100966981
Informations de publication
Date de publication:
29 10 2021
29 10 2021
Historique:
received:
04
04
2021
accepted:
01
10
2021
entrez:
30
10
2021
pubmed:
31
10
2021
medline:
14
1
2022
Statut:
epublish
Résumé
Formalin-fixed paraffin embedded (FFPE) tissues may provide an exciting resource to study microbial associations in human disease, but the use of these low biomass specimens remains challenging. We aimed to reduce unintentional bacterial interference in molecular analysis of FFPE tissues and investigated the feasibility of conducting quantitative polymerase chain reaction (qPCR) and 16S rRNA amplicon sequencing using 14 colorectal cancer, 14 normal adjacent and 13 healthy control tissues. Bacterial contaminants from the laboratory environment and the co-extraction of human DNA can affect bacterial analysis. The application of undiluted template improves bacterial DNA amplification, allowing the detection of specific bacterial markers (Escherichia coli and Faecalibacterium prausnitzii) by qPCR. Nested and non-nested PCR-based 16S rRNA amplicon sequencing approaches were employed, showing that bacterial communities of tissues and paired paraffin controls cluster separately at genus level on weighted Unifrac in both non-nested (R2 = 0.045; Pr(> F) = 0.053) and nested (R2 = 0.299; Pr(> F) = 0.001) PCR datasets. Nevertheless, considerable overlap of bacterial genera within tissues was seen with paraffin, DNA extraction negatives (non-nested PCR) or PCR negatives (nested PCR). Following mathematical decontamination, no differences in α- and β diversity were found between tumor, normal adjacent and control tissues. Bacterial marker analysis by qPCR seems feasible using non-normalized template, but 16S rRNA amplicon sequencing remains challenging. Critical evaluation of laboratory procedures and incorporation of positive and negative controls for bacterial analysis of FFPE tissues are essential for quality control and to account for bacterial contaminants.
Sections du résumé
BACKGROUND
Formalin-fixed paraffin embedded (FFPE) tissues may provide an exciting resource to study microbial associations in human disease, but the use of these low biomass specimens remains challenging. We aimed to reduce unintentional bacterial interference in molecular analysis of FFPE tissues and investigated the feasibility of conducting quantitative polymerase chain reaction (qPCR) and 16S rRNA amplicon sequencing using 14 colorectal cancer, 14 normal adjacent and 13 healthy control tissues.
RESULTS
Bacterial contaminants from the laboratory environment and the co-extraction of human DNA can affect bacterial analysis. The application of undiluted template improves bacterial DNA amplification, allowing the detection of specific bacterial markers (Escherichia coli and Faecalibacterium prausnitzii) by qPCR. Nested and non-nested PCR-based 16S rRNA amplicon sequencing approaches were employed, showing that bacterial communities of tissues and paired paraffin controls cluster separately at genus level on weighted Unifrac in both non-nested (R2 = 0.045; Pr(> F) = 0.053) and nested (R2 = 0.299; Pr(> F) = 0.001) PCR datasets. Nevertheless, considerable overlap of bacterial genera within tissues was seen with paraffin, DNA extraction negatives (non-nested PCR) or PCR negatives (nested PCR). Following mathematical decontamination, no differences in α- and β diversity were found between tumor, normal adjacent and control tissues.
CONCLUSIONS
Bacterial marker analysis by qPCR seems feasible using non-normalized template, but 16S rRNA amplicon sequencing remains challenging. Critical evaluation of laboratory procedures and incorporation of positive and negative controls for bacterial analysis of FFPE tissues are essential for quality control and to account for bacterial contaminants.
Identifiants
pubmed: 34715774
doi: 10.1186/s12866-021-02359-z
pii: 10.1186/s12866-021-02359-z
pmc: PMC8555202
doi:
Substances chimiques
DNA, Bacterial
0
RNA, Ribosomal, 16S
0
Formaldehyde
1HG84L3525
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
297Informations de copyright
© 2021. The Author(s).
Références
J Microbiol Methods. 2006 Jul;66(1):21-31
pubmed: 16305811
Oncol Lett. 2010 May;1(3):555-558
pubmed: 22966342
Anal Biochem. 2009 Dec 15;395(2):265-7
pubmed: 19698695
Biochem Biophys Res Commun. 1987 Feb 13;142(3):710-6
pubmed: 3548717
Nat Microbiol. 2018 Aug;3(8):851-853
pubmed: 30046175
Gut Pathog. 2016 May 26;8:24
pubmed: 27239228
Nutr Rev. 2012 Aug;70 Suppl 1:S38-44
pubmed: 22861806
BMC Microbiol. 2019 Mar 4;19(1):52
pubmed: 30832576
mSystems. 2019 Jun 4;4(4):
pubmed: 31164452
Pediatr Infect Dis J. 2012 Apr;31(4):413-4
pubmed: 22173144
Biometrics. 2006 Mar;62(1):245-53
pubmed: 16542252
Int J Mol Sci. 2020 Feb 07;21(3):
pubmed: 32046034
Nature. 2011 May 12;473(7346):174-80
pubmed: 21508958
PLoS One. 2013 Apr 22;8(4):e61217
pubmed: 23630581
Pathology. 2007 Jun;39(3):345-8
pubmed: 17558863
Inflamm Bowel Dis. 2016 Jan;22(1):28-41
pubmed: 26595550
mSystems. 2019 Feb 19;4(1):
pubmed: 30801029
Asian Pac J Cancer Prev. 2007 Jan-Mar;8(1):55-9
pubmed: 17477772
Front Genet. 2020 Jan 23;10:1366
pubmed: 32117417
Nature. 2020 Apr;580(7802):269-273
pubmed: 32106218
BMC Cancer. 2019 Apr 29;19(1):399
pubmed: 31035942
Sci Rep. 2021 Jan 11;11(1):391
pubmed: 33432015
Histol Histopathol. 2011 Jun;26(6):797-810
pubmed: 21472693
Biopreserv Biobank. 2013 Apr;11(2):101-6
pubmed: 24845430
J Gastroenterol Hepatol. 2008 Aug;23(8 Pt 1):1298-303
pubmed: 18624900
Microbiome. 2018 Dec 17;6(1):226
pubmed: 30558668
Bioinformatics. 2010 Oct 1;26(19):2460-1
pubmed: 20709691
Pathol Res Pract. 2014 Mar;210(3):142-6
pubmed: 24355442
Nucleic Acids Res. 2013 Jan;41(Database issue):D590-6
pubmed: 23193283
Lett Appl Microbiol. 2019 Jan;68(1):2-8
pubmed: 30383890
BMC Microbiol. 2011 Apr 12;11:73
pubmed: 21486476
mBio. 2020 Jun 9;11(3):
pubmed: 32518181
Expert Rev Mol Diagn. 2013 Apr;13(3):271-82
pubmed: 23570405
PLoS One. 2012;7(4):e34683
pubmed: 22514653
PLoS One. 2007 Jun 20;2(6):e537
pubmed: 17579711
Forensic Sci Int. 2010 Jan 30;194(1-3):e25-8
pubmed: 19781880
J Clin Virol. 2016 Jul;80:36-9
pubmed: 27148635
PLoS One. 2019 Jun 13;14(6):e0218257
pubmed: 31194836
F1000Res. 2016 Jul 22;5:1791
pubmed: 30918626
Science. 2012 Oct 5;338(6103):120-3
pubmed: 22903521
J Surg Res. 2017 Dec;220:125-132
pubmed: 29180174
BMC Biol. 2014 Nov 12;12:87
pubmed: 25387460
N Engl J Med. 2016 Dec 15;375(24):2369-2379
pubmed: 27974040