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    Viroma

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    The study analyzed the virome of human gut samples from two experiments: 1) COMBO analyzed correlations between diet, bacteria, and viruses using questionnaires and stool samples from 97 subjects. Several bacterial populations correlated with diet components. 2) CAFE controlled feeding experiments on 10 subjects undergoing high fat vs fiber diets for 10 days while hospitalized. Bacterial and viral populations changed within 24 hours and differed between diets. The virome was characterized from stool samples, finding 7,000 viral contigs and interpersonal variation dominated over dietary effects.

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    Viroma

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    The study analyzed the virome of human gut samples from two experiments: 1) COMBO analyzed correlations between diet, bacteria, and viruses using questionnaires and stool samples from 97 subjects. Several bacterial populations correlated with diet components. 2) CAFE controlled feeding experiments on 10 subjects undergoing high fat vs fiber diets for 10 days while hospitalized. Bacterial and viral populations changed within 24 hours and differed between diets. The virome was characterized from stool samples, finding 7,000 viral contigs and interpersonal variation dominated over dietary effects.

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    The study analyzed the virome of human gut samples from two experiments: 1) COMBO analyzed correlations between diet, bacteria, and viruses using questionnaires and stool samples from 97 subjects. Several bacterial populations correlated with diet components. 2) CAFE controlled feeding experiments on 10 subjects undergoing high fat vs fiber diets for 10 days while hospitalized. Bacterial and viral populations changed within 24 hours and differed between diets. The virome was characterized from stool samples, finding 7,000 viral contigs and interpersonal variation dominated over dietary effects.

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    © All Rights Reserved
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    14 views22 pages

    Viroma

    Uploaded by

    webcentrobio
    The study analyzed the virome of human gut samples from two experiments: 1) COMBO analyzed correlations between diet, bacteria, and viruses using questionnaires and stool samples from 97 subjects. Several bacterial populations correlated with diet components. 2) CAFE controlled feeding experiments on 10 subjects undergoing high fat vs fiber diets for 10 days while hospitalized. Bacterial and viral populations changed within 24 hours and differed between diets. The virome was characterized from stool samples, finding 7,000 viral contigs and interpersonal variation dominated over dietary effects.

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    The virome of the human gut:

    metagenomic analysis of changes


    associated with diet

    James Lewis
    Gary Wu
    Frederic Bushman

    Diet, Genetic Factors, and the Gut Microbiome in Crohn’s Disease

    University of Pennsylvania
    Diet, Genetic Factors, and the Gut
    Microbiome in Crohn’s Disease

    COMBO: Cross-Sectional Study of Diet and


    Stool Microbiome

    CAFE: Controlled Feeding Experiment

    Study virome under controlled feeding


    COMBO
    97 subjects, assess diet with food frequency
    questionnaire, 24 hr. recall questionnaire
    454 tag sequencing of 16S regions

    Bacteria

    *p<0.05
    Food groups **p<0.01
    ***p<0.001
    diet components called at 25% FDR
    COMBO (selected)
    BMI Fiber Fat %Calories Actinobacteria

    Proteobacteria

    Bacteroidetes

    Firmicutes

    ***p<0.001
    **p<0.01
    *p<0.05
    Conclusions from COMBO
    •At an FDR of 25%, ~30-40 nutrients correlated with changes
    in bacterial populations

    •Correlations between food groups and Pyla detected, but


    considerable divergence among deeper taxa

    •Correlated effects on microbiome among BMI, dietary fat,


    and percent calories from saturated fatty acids

    •Though effects were significant, measured dietary effects


    accounted for a small fraction of the total variation among
    subjects

    Follow up in CAFÉ: 1) fat versus fiber, 2) total calories


    CAFE1: Controlled Feeding Experiment

    • 10 healthy volunteers
    • Randomized to high fat vs. low fat diet
    • 10 day inpatient stay with same meals each day
    • Caloric intake adjusted to maintain current weight
    • Daily stool sample collection
    • Rectal biopsies on days 1 and 10
    • Sitz marker study to assess transit time
    • Sequencing: 16S tags, shot-gun metagenomic
    sequencing of total DNA and viral DNA, targeted
    analysis of Archaea and Eukarya
    Longitudinal analysis of microbiome under controlled feeding

    Unweighted
    Unifrac

    Graphic from
    QIIME
    Conclusions from CAFÉ 1 and 2
    •Inter-individual variation predominates.

    •Bacterial populations change within 24 hours of initiating


    controlled diet.

    •High fat versus fiber has detectable effect (CAFE1),


    increased calories has detectable effect (CAFE2).

    •Considerable longitudinal drift during stay in hospital in


    all groups. Specific vitamins? Hospital environment?
    Approaches to virome analysis

    Multiple possible goals:

    •Characterize overall viral communities

    •Hunt for new viruses linked to disease

    •Characterize populations of a specific virus


    Viral Analysis on CAFE1 samples
    • Purification of viral particles: filtration, CsCl gradient, DNAse digestion.
    • Quantitative recovery of phage λ analyzed as a control
    • Greatly reduced 16S rDNA
    • Multiple displacement amplification
    • Shot-gun sequencing, 454 Titanium, 992,309 reads, median length 380 nt
    PID Day 1 2 3 4 5 6 7 8 Diet

    2011 X X Low
    Fat Sybr gold
    2012 X X X Low
    Fat
    staining
    2016 X X X X High
    Fat
    2020 X X X X Low
    Fat
    2019 X X High
    Fat
    1013 X Ad lib
    1010 phage per gram of stool

    10
    Sam Minot et al.
    Assembly of viral sequence reads

    • Newbler assembler
    40bp overlap
    90% sequence identity
    • 7,175 contigs >500 bp
    • 86.6% of reads in contigs
    • Custom code to allow circular
    assembly
    • PHACCS: median species
    richness 44 (range=19-785)

    11
    12
    Gut virome characterization

    Also found 22 CRISPR arrays, one example of CRISPR spacer targeting another virus in
    the same individual 14
    Comparative metagenomics:
    viruses are parasites
    Lysogeny
    Interpersonal variation

    • Rows: samples
    • Columns: contigs

    • Clustering by
    individual

    17
    Variation associated with
    subject and diet

    • People are more


    similar to
    themselves
    • People on the
    same diet become
    more similar over
    time
    • Specify the contigs
    involved
    18
    P values by label permutation
    Procrustes analysis: covariation of
    host and viral communities
    What determines phage
    abundance?
    • Abundance of host?
    • Lysogenic induction?

    Phage
    abundance

    Host abundance

    In some cases the abundance of phage does not correlate with apparent
    abundance of host: possibly indicative of differential induction?
    Summary

    • Variation in both bacterial and viral communities


    dominated by interpersonal variation
    • Dietary intervention associated with altered
    composition of both bacterial and viral communities
    • Metagenomic analysis over six subjects yielded 7000
    viral contigs; 19-785 types per sample (minimum
    estimate)
    • Both expected and novel functionality, one example
    being viral CRISPRs targeting other viruses
    Credits
    UPenn/CHOP Group Collaborators
    Gary Wu* Bob Baldassano Rob Knight
    James Lewis* Sue Keilbaugh Catherine Lozupone
    Frederic Bushman* Ying-Yu Chen Dan Knights
    Sam Minot Lisa Nessel
    Emily Charlson Jun Chen
    Christian Hoffmann Hongzhi Li
    Scott Sherrill-Mix
    Serena Dollive
    * co-PIs
    Kyle Bittinger
    Rohini Sinha
    Jennifer Hwang
    Rebecca Custers-Allen
    Stephanie Grunberg

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