DOI: 10.5061/dryad.z8w9ghxrf
title: 16S rRNA gene sequencing data from: Breastmilk IgG engages the neonatal immune system to instruct immune responses to gut antigens

description: Maternal antibodies fundamentally regulate gut immunity in the developing
 infant, yet the mechanisms underlying this process remain elusive. Here,
 we show that maternal IgG, ingested in the first week of life, restrains
 microbiota-dependent adaptive immune responses weeks later, following
 weaning. Antibodies are key regulators of gut microbiota diversity and
 composition, prompting us to explore whether alterations in microbiome
 assembly correlated with immune dysregulation in maternal
 antibody-deficient offspring. 16S rRNA gene sequencing of ileal and
 colonic contents did not reveal substantial differences in the diversity
 of microbes in each sample (alpha diversity) nor between-group distances
 (beta diversity) between age-matched offspring of µMT+/- or µMT-/-
 littermate dams and B6 sires. However, the abundance of a small subset of
 taxa differed significantly between p7 offspring of µMT+/- and µMT-/-
 dams, and this variation increased with age. Thus, the absence of all
 breastmilk antibodies correlates with minor alterations in the assembly of
 the postnatal microbiome. Analysis of p20 progeny of µMT-/- dams and B6
 sires fed sIgG or BSA during the first week of life similarly failed to
 reveal significant differences in alpha or beta diversity as a function of
 feeding. Instead, litter was a driving factor. Indeed, the provision of
 IgG resulted in the differential abundance of only a single ileal taxon
 and two colonic taxa, which were distinct from those identified in p21
 offspring of µMT+/- and µMT-/- dams. The similarity in microbiota
 community structure between IgG and BSA-fed littermates indicates that
 breastmilk IgG does not significantly alter the composition of the
 developing microbiome.
description: Amplification and MiSeq Illumina Sequencing of the V4 region of
 the 16S gene was performed by the Alkek Center for Metagenomics and
 Microbiome Research (CMMR) at Baylor College of Medicine. Reads were
 demultiplexed, trimmed and quality checked with bbduk. Paired-end reads
 were then merged using bbmap and run through the MaLiAmPi workflow to
 generate Amplicon Sequence Variants (ASVs) using DADA2. The Bayesian
 classifier Pplacer was used to estimate taxonomic classification of ASVs
 using phylogenetic placement with a likelihood cutoff of 90%. Alpha
 diversity estimates were generated by MaLiAmPi and Bray-Curtis
 dissimilarity was calculated using the R package
 <em>vegan</em> (v2.6-4) to determine beta diversity estimates.
 A pseudocount was applied prior to calculating differences in the relative
 abundance of taxonomic groups and ASVs with fewer than 10 reads across all
 samples were removed from the dataset. Differential abundance was
 determined using DESeq2, and data was normalized using the “poscounts”
 size estimator.
description: # Breastmilk IgG engages the neonatal immune system to instruct immune
 responses to gut antigens #### Dataset Title 16S rRNA Gene Sequencing Data
 from: Breastmilk IgG engages the neonatal immune system to instruct immune
 responses to gut antigens #### Principal Investigator Information Name:
 Meghan Koch ORCID: 0000-0002-9539-4027 Institution: Fred Hutchinson Cancer
 Center Address: 1100 N. Fairview Ave. Seattle, WA 98109 Email:
 [mkoch@fredhutch.org](mailto:mkoch@fredhutch.org) #### Author Information
 Name: Meera Shenoy ORCID: 0000-0002-3986-1321 Institution: Singapore
 Immunology Network, Agency for Science, Technology, and Research Address:
 8a Biomedical Grove, Singapore 138648 Email:
 [Meera_Shenoy@immunol.a-star.edu.sg](mailto:Meera_Shenoy@immunol.a-star.edu.sg) Dates of Data Collection 2016-11-16: Maternal Antibody Sufficient vs. Deficient Version 2 2017-10-26: Maternal Antibody Sufficient vs. Deficient Version 3 2022-05-05: IgG vs. BSA fed #### Geographic Location of Data Collection: Maternal Antibody Sufficient vs. Deficient Versions 2 and 3: Berkeley, CA IgG vs. BSA fed: Seattle, WA #### DATA PROCESSING Amplification and MiSeq Illumina sequencing were performed by the Alkek Center for Metagenomics and Microbiome Research at Baylor College of Medicine. The V4 region of the 16S rRNA gene was amplified using 515F and 806R primers. Reads were demultiplexed, trimmed, and quality checked with bbduk. The 250bp paired-end reads were then merged using bbmap and run through the MaLiAmPi workflow to generate Amplicon Sequence Variants (ASVs) using DADA2. The Bayesian classifier Pplacer was used to estimate taxonomic classification of ASVs using phylogenetic placement with a likelihood cutoff of 90%. Sequences that could not be assigned unambiguously to a single genus were instead assigned at a higher taxonomic level. #### EXPERIMENT SUMMARY **Maternal Antibody Sufficient vs. Deficient Experiment (matAb-sufficient vs. deficient**) We bred µMT-knockout C57Bl/6 dams that lacked antibodies with wild-type sires to produce µMT-heterozygous offspring that received no maternal antibodies yet were capable of producing their own antibodies. We collected intestinal samples from these mice as well as from mice born to µMT-heterozygous C57Bl/6 dams and C57Bl/6 wild-type sires, and sequenced the 16S rRNA gene to characterize the composition of the microbiome in the two groups. µMT-heterozygous and µMT-knockout dams used for experiments were littermates and were cohoused before breeding to promote homogenization of the microbiome. **IgG Feed Experiment (IgG_vs_BSA-fed)** This dataset contains 16S rRNA sequencing reads from pups lacking maternal antibodies that were fed 10 µg of either IgG purified from the serum of specific-pathogen-free (SPF) mice or the protein bovine serum albumin (BSA; control) once daily during the first week of life for a total of eight feedings. #### DIRECTORY OVERVIEW Each experiment directory contains * .csv metadata file with information about each sample using the data-specific abbreviations described below. * A directory containing demultiplexed compressed FASTQ files containing forward and reverse paired-end reads for each sample. * Forward reads filenames end with either *1.fq.gz* or *R1.fastq.gz*. * Reverse reads filenames end with either *2.fq.gz* or *R2.fastq.gz.* * Directory containing the ASV counts table, taxa tables (counts and relative abundance), ASV sequences, and ASV classification. #### TABULAR METADATA FILE DETAILS Samples are in rows. Each column includes information about each sample as described below. #### matAb-sufficient\_vs\_deficient\_metadata.csv **sampleID** Sample IDs include information about the dam genotype, timepoint, cage ID, mouse number, and sampling location in that order. The sample IDs use the abbreviations described for each column header below. For example, sample Het14A_1_col is from a maternal antibody sufficient mouse, from the day 14 timepoint, cohort A, is mouse #1 from that litter and timepoint, and collected from the colon contents. **damGeno (dam genotype)** Indicates maternal antibody status. * Het: Maternal antibody sufficient – mouse was born to and nursed by a C57Bl/6 dam able to produce antibodies (heterozygous for the µMT gene) * KO: maternal antibody deficient – mouse was born to and nursed by a C57Bl/6 dam not able to produce antibodies (homozygous KO for the µMT gene) **timepoint** Time point of sample collection (postnatal days). * 7 * 14 * 21 **cohort** Groups of litters that were born on or around the same day. * Cohorts A and B are from Version 2. * Cohorts C and D are from Version 3. **number** Each mouse with identical parameters was assigned a unique number. Missing data is represented with "NA". **location1** Site of tissue/intestinal contents sampled. intestine samples * col: contents of the distal colon * ile: contents of the ileum tissue samples * lgwall: distal colon wall * smwall: ileal wall **version** Version 2 (v2): experiment performed in 2016 Version 3 (V3): repeat experiment performed in 2017 #### IgG\_vs\_BSA-fed\_metadata.csv **sampleID** Sample IDs include the cage ID, mouse number, and sampling site (C=colon lumen, S=ileum lumen). Sample IDs include information about the dam genotype, timepoint, cage ID, mouse number, and sampling location in that order. The sample IDs use the abbreviations described for each column header below. For example, sample Het14A_1_col is from a maternal antibody sufficient mouse, from the day 14 timepoint, cohort A, is mouse #1 from that litter and timepoint, and collected from the colon contents. **feed** Indicates whether the mouse was fed IgG or BSA (control). * IgG10: 10 µg IgG * BSA10: 10 µg BSA * na: The dams were not fed IgG or BSA as part of this experiment. This is indicated by "na" in this column. **location1** * ColonLumen: Samples are from the contents of the colon lumen. * Ileum Lumen: Samples are from the contents of the ileal lumen. * Feces: Fecal pellets, collected from the dams only. **Cage** Indicates which cage the mouse was housed in. Mice with the same cage number are the same age and from the same litter. **Sex** * f: female * m: male **AgeAtSac** The postnatal day on which the mouse was sacrificed and tissues collected. **type** Indicates whether the sample is from a pup or a dam. Fecal samples from the three dams were collected to compare with the pups’ microbiome. * pup: experimental mouse (offspring of ADam, BDam, and EDam) * Adam: from cage 739A * DDam: dam from cage 739D * EDam: dam from cage 739E #### FASTQ FILENAMES Each filename includes information about the sample as described below. #### matAb-sufficient\_vs\_deficient\_raw\_demux\_reads\_V2\_V3 * Versions 2 and 3 were processed at different times and have slight differences in their naming schemes. For more information about each category, please see the TABULAR METADATA FILE DETAILS. ##### Version 2 files * MatAb: experiment - Maternal antibody sufficient vs. deficient * v2: Version 2 * Intestine/Tissue: intestine = intestinal contents, tissue = intestinal wall * Barton: PI of project - Greg Barton, University of California, Berkeley * Het/KO: Het = maternal antibody sufficient, KO = maternal antibody deficient * 7/14/21: timepoint of specimen collection (postnatal day) * A/B: experimental cohort * 1/2/3/4/5/6: mouse number * col/ile/sm_w/lg_w: col = colon, ile = ileum, sm_wall = small intestine wall, lg_w = large intestine wall * R1/R2: R1 = forward paired-end reads, R2 = reverse paired-end reads ##### Version 3 files * 102617: date the files were submitted for sequencing - Oct. 26, 2017 * MatAb: experiment - Maternal antibody sufficient vs. deficient * V3: Version 3 * Barton: PI - Greg Barton, UCB * Het/KO: Het = maternal antibody sufficient, KO = maternal antibody deficient * 7/14/21: timepoint of specimen collection * C/D: experimental cohort * col/ile/sm_w/lg_w: col = colon, ile = ileum, sm_wall = small intestine wall, lg_w = large intestine wall * R1/R2: R1 = forward paired-end reads, R2 = reverse paired-end reads #### IgG vs. BSA-fed * Koch: PI - Meghan Koch, Fred Hutchinson Cancer Center * A/D/E: litter ID * 1/2/3/4/5/6: mouse number * C/S: C = colon contents S = small intestine (ileum) contents * 1/2: 1 = forward reads, 2 = reverse reads #### SPECIMEN IDS IN ASV TABLES AND CLASSIFICATION FILES Specimen IDs are detailed below. #### matAb-sufficient\_vs\_deficient ##### Version 2 files * v2: Version 2 * Intestine/Tissue: Intestine = Intestinal wall, Tissue = Tissue contents * KO/Het: maternal antibody sufficient or deficient * 7/14/21: timepoint of collection (postnatal day) * A/B: A = cohort A, B = cohort B * 1/2/3/4/5/6: mouse number * col/ile/sm_w/lg_w: col = colon, ile = ileum, sm_wall = small intestine wall, lg_w = large intestine wall ##### Version 3 files * V3: Version 3 * KO/Het: maternal antibody sufficient or deficient * C/D: C = cohort C, D = cohort D * 7/14/21: timepoint of collection (postnatal day) * 1/2/3/4/5/6: mouse number * col/ile/smwall/lgwall: col = colon contents, ile = ileum contents, smwall = small intestine wall, lgwall = large intestine wall #### IgG\_vs\_BSA-fed * A/D/E: litter * 1/2/3/4/5/6: mouse number * C/S: C = colon contents, S = ileum contents (small wall and large wall samples from this experiment were not included in this analysis) #### FILE STRUCTURE * IgG_vs_BSA-fed * IgG_vs_BSA-fed_metadata.csv * IgG_vs_BSA-fed_raw_demux_reads/ * Koch_D1C_1.fq.gz * Koch_D1C_2.fq.gz * Koch_D1S_1.fq.gz * Koch_D1S_2.fq.gz * … * ASV_tables_and_classification * dada2.combined.seqtabs.nochimera.csv *(ASV table: ASV counts for each sample)* * dada2.sv.fasta *(sequences of each ASV)* * sv_taxonomy.csv *(taxonomic classification for each ASV)* * tables * taxon_wide_nreads.class.csv *(Taxa tables: number of each taxon for each sample)* * taxon_wide_nreads.family.csv * taxon_wide_nreads.genus.csv * taxon_wide_nreads.order.csv * taxon_wide_nreads.phylum.csv * taxon_wide_nreads.species.csv * taxon_wide_ra.class.csv *(RA tables: relative abundance of each taxon for each sample)* * taxon_wide_ra.family.csv * taxon_wide_ra.genus.csv * taxon_wide_ra.order.csv * taxon_wide_ra.phylum.csv * taxon_wide_ra.species.csv * matAb-sufficient_vs_deficient * matAb-sufficient_vs_deficient_metadata.csv * matAb-sufficient_vs_deficient_raw_demux_reads_V2_V3/ * 102617-MatAb-V3_Barton_Het14C1col_R1.fastq.gz * 102617-MatAb-V3_Barton_Het14C1col_R2.fastq.gz * 102617-MatAb-V3_Barton_Het14C1ile_R1.fastq.gz * 102617-MatAb-V3_Barton_Het14C1ile_R2.fastq.gz * … * ASV_tables_and_classification * dada2.combined.seqtabs.nochimera.csv *(ASV table: ASV counts for each sample)* * dada2.sv.fasta *(sequences of each ASV)* * sv_taxonomy.csv *(taxonomic classification for each ASV)* * tables/ * taxon_wide_nreads.class.csv *(Taxa tables: number of each taxon for each sample)* * taxon_wide_nreads.family.csv * taxon_wide_nreads.genus.csv * taxon_wide_nreads.order.csv * taxon_wide_nreads.phylum.csv * taxon_wide_nreads.species.csv * taxon_wide_ra.class.csv *(RA tables: relative abundance of each taxon for each sample)* * taxon_wide_ra.family.csv * taxon_wide_ra.genus.csv * taxon_wide_ra.order.csv * taxon_wide_ra.phylum.csv * taxon_wide_ra.species.csv