Sample Processing

Branchpoint Biosciences qPCR Sample Processing

Performed on extracted DNA (preferred).

Each sample undergoes:

(confirmational) fluorometric DNA quantification
quantitative PCR targeting either -
1. 12 members of the Faecalibacterium prausnitzii species complex
2. 7 members of the Fusobacterium nucleatum species complex
3. 4 members of the Akkermansia muciniphila species complex

Data returned in simple Excel spreadsheet with quantitative values calculated as genome equivalents per ng DNA. For less than 81 samples, data delivered within 48 hours of receipt of samples.

Pricing

Pricing on request - discounts offered for first-time clients and volume contracts.

To prepare your DNA for qPCR, follow these steps:

For orders ≤54 samples, we accept 8-strip PCR tubes, 1.5 mL Eppendorf tubes, or 96-well plate. Be sure to clearly label each tube with the corresponding sample number. For orders ≥54 samples, we recommend a 96-well plate with the samples ordered vertically (A1 to H1).

Prepare template DNA for qPCR using the (strongly) recommended total DNA values listed below.

Target	Minimum DNA Concentration	Total DNA Required	Minimum volume
Faecalibacterium prausnitzii	20 ng/μL	400 ng	20 μL
Fusobacterium nucleatum	20 ng/μL	300 ng	15 μL
Akkermansia muciniphila	20 ng/μL	200 ng	10 μL

Additional service fee(s). Branchpoint Biosciences can extract DNA from biological samples (excluding tissue) using our standard DNA extraction protocol for an additional fee for up to 27 samples.

For more information describing species complexes offered as part of this service, please select from the following options below:

Faecalibacterium prausnitzii

Health Summary. Faecalibacterium prausnitzii and the species within this complex are some of the most abundant commensal bacteria in the human gut. F. prausnitzii has garnered significant interest due to its role in the production of butyrate and other short chain fatty acids as part of the fermentation of dietary fibers in the gut. Butyrate and short chain fatty acids have a role in promoting a healthy gut and decreasing inflammation. No longer considered a single species, research by De Filippis et al. (2020) (and confirmed by others, including Branchpoint Biosciences) shows that most individuals can harbor between 1 to 12 different F. prausnitzii-related species.

Target Designation. Branchpoint Biosciences anchors our species designations using the Genome Taxonomy Database (GTDB). The GTDB is based on evolutionary distance and nucleotide identity, offering clear delineations between species and higher taxonomic ranks. In their 2022 release (R207), Faecalibacterium prausnitzii was split into 12 species. Since, efforts have shown that type strains of F. prausnitzii available within public repositories constitute representatives of these new species and have received official recognition from the International Journal of Systematic and Evolutionary Microbiology (IJSEM), the governing body of microbiology systematics. Each of the 12 targets on this panel were once lumped together as F. prausnitzii and this targeted panel allows you to disentangle that signal.

Alternative names.

Faecalibacterium prausnitzii_C has been renamed F. duncaniae.

Faecalibacterium prausnitzii_G has been renamed F. longum.

Faecalibacterium prausnitzii_H has been renamed F. hattorri.

Branchpoint Biosciences Research. We describe the marked differences between qPCR detection of F. prausnitzii-related species and bioinformatic methods in our blog post about metagenomic analysis and in greater detail within our first company manuscript.

Our reference strain for Faecalibacterium prausnitzii is represented by Faecalibacterium prausnitzii DSM 107838 (strain designation APC918/95b).

GTDB Designation (R207)	GTDB Designation (R214)	Branchpoint Reference Strain
Faecalibacterium prausnitzii	Faecalibacterium prausnitzii	DSM 107838
Faecalibacterium prausnitzii_A	Faecalibacterium prausnitzii_A	n/a
Faecalibacterium prausnitzii_C	Faecalibacterium duncaniae	DSM 17677
Faecalibacterium prausnitzii_D	Faecalibacterium prausnitzii_D	n/a
Faecalibacterium prausnitzii_E	Faecalibacterium prausnitzii_E	n/a
Faecalibacterium prausnitzii_F	Faecalibacterium prausnitzii_F	n/a
Faecalibacterium prausnitzii_G	Faecalibacterium longum	DSM 103432
Faecalibacterium prausnitzii_H	Faecalibacterium hattorri	DSM 107841
Faecalibacterium prausnitzii_I	Faecalibacterium prausnitzii_I	n/a
Faecalibacterium prausnitzii_J	Faecalibacterium prausnitzii_J	n/a
Faecalibacterium prausnitzii_L	Faecalibacterium prausnitzii_L	n/a
Faecalibacterium prausnitzii_M	Faecalibacterium prausnitzii_M	n/a

Fusobacterium nucleatum

Health Summary. Fusobacterium nucleatum is considered a commensal organism within the oral cavity with a role in periodontal disease. Though considered a commensal organism in the mouth, F. nucleatum has been linked with numerous conditions and diseases in other body sites. One important association is between F. nucleatum and colorectal cancer. Recent findings have implicated F. nucleatum in promoting a pro-inflammatory environment which may contribute to tumor formation and growth. F. nucleatum canonical represents a species complex and was commonly split into four subspecies.

Target Designation. Branchpoint Biosciences anchors our species designations using the Genome Taxonomy Database (GTDB). The GTDB is based on evolutionary distance and nucleotide identity, offering clear delineations between species and higher taxonomic ranks. In their 2022 release (R207), Fusobacterium nucleatum was split into multiple species. The four subspecies - F. nucleatum subsp. nucleatum, F. nucleatum subsp. animalis, F. nucleatum subsp. vincentii, and F. nucleatum subsp. polymorphum - were renamed as four distinct species. Each of the 7 targets on this panel were once lumped together as F. nucleatum and this targeted panel allows you to disentangle that signal.

Alternative names.

nucleatum subsp. nucleatum has the new designation F. nucleatum.
nucleatum subsp. animalis has the new designation F. animalis.
nucleatum subsp. vincentii has the new designation F. vincentii.
nucleatum subsp. polymorphum has the new designation F. polymorphum.

Branchpoint Biosciences Research. We discuss in a blog post on how legacy qPCR primers in the literature may lead to additional confusion in identifying F. nucleatum and its subspecies in human samples.

Our reference strain for Fusobacterium nucleatum is represented by Fusobacterium nucleatum subsp. nucleatum DSM 15643 (strain designation: VPI 4355; additional repository collections: ATCC 25586, CCUG 32989, CIP 101130, JCM 8532, LMG 13131).

GTDB Designation (R207)	GTDB Designation (R214)	Branchpoint Reference Strain
Fusobacterium animalis	Fusobacterium animalis	DSM 19679
Fusobacterium canifelinum	Fusobacterium canifelinum	DSM 15542
Fusobacterium nucleatum	Fusobacterium nucleatum	DSM 15643
Fusobacterium nucleatum_E	Fusobacterium nucleatum_E	n/a
Fusobacterium nucleatum_J	Fusobacterium nucleatum_J	n/a
Fusobacterium polymorphum	Fusobacterium polymorphum	DSM 20482
Fusobacterium vincentii	Fusobacterium vincentii	DSM 19507

Akkermansia muciniphila

Health Summary. Akkermansia muciniphila is considered a commensal organism within the human gut. Only identified in 2004, A. muciniphila plays an important role in maintaining a healthy balance of the intestinal mucosal lining. When in homeostasis with its human host, A. muciniphila can prevent other harmful bacteria from colonizing and degrading the mucosal lining. A. muciniphila has been identified as a potential probiotic which may have impacts on metabolic diseases.

Target Designation. Branchpoint Biosciences anchors our species designations using the Genome Taxonomy Database (GTDB). The GTDB is based on evolutionary distance and nucleotide identity, offering clear delineations between species and higher taxonomic ranks. In their 2022 release (R207), Akkermansia muciniphila was split into multiple species. Outside of the A. muciniphila type strain, the other species are uncultivated and identified through environmental genomic data alone. Each of the 4 targets on this panel were once lumped together as A. muciniphila andthis targeted panel allows you to disentangle that signal.

Alternative names.

Not available.

Branchpoint Biosciences Research. Our reference strain for Akkermansia muciniphila is represented by Akkermansia muciniphila DSM 26127 (strain designation: YL44).

GTDB Designation (R207)	GTDB Designation (R214)	Branchpoint Reference Strain
Akkermansia muciniphila	Akkermansia muciniphila	DSM 26127
Akkermansia muciniphila_A	Akkermansia muciniphila_A	n/a
Akkermansia muciniphila_B	Akkermansia muciniphila_B	n/a
Akkermansia muciniphila_C	Akkermansia muciniphila_C	n/a
Akkermansia muciniphila_D	Akkermansia muciniphila_D	n/a