Oct 29, 2024
IgA Metagenomic Immunoglobulin Sequencing (MIG-Seq)
- Matthew R Olm1,
- Sean Spencer1
- 1Stanford University
Protocol Citation: Matthew R Olm, Sean Spencer 2024. IgA Metagenomic Immunoglobulin Sequencing (MIG-Seq). protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8pk6dg2w/v1
Manuscript citation:
https://doi.org/10.1101/2023.11.21.568153
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 21, 2023
Last Modified: October 29, 2024
Protocol Integer ID: 91304
Keywords: IgA, IgA-SEQ, MIG-Seq
Funders Acknowledgement:
Matthew Olm
Grant ID: F32DK128865
Matthew Olm
Grant ID: T32 AI007328-30
Sean Spencer
Grant ID: The Colleen and Robert D. Hass fund
Sean Spencer
Grant ID: T32DK007056
Sean Spencer
Grant ID: K08DK134856
Abstract
IgA, the most highly produced human antibody, is continually secreted into the gut to shape the intestinal microbiota. Methodological limitations have critically hindered defining which microbial strains are targeted by IgA and why. Here, we develop a new technique, Metagenomic Immunoglobulin Sequencing (MIG-Seq), and use it to determine IgA coating levels for thousands of gut microbiome strains in healthy humans. We find that microbes associated with both health and disease have higher levels of coating, and that microbial genes are highly predictive of IgA binding levels, with mucus degradation genes especially correlated with high binding. We find a significant reduction in replication rates among microbes bound by IgA, and demonstrate that IgA binding is more correlated with host immune status than traditional microbial abundance measures. This study introduces a powerful technique for assessing strain-level IgA binding in human stool, paving the way for deeper understanding of IgA-based host microbe interactions.
Protocol materials
EasySep™ APC Positive Selection Kit IISTEMCELL Technologies Inc.Catalog #17681
In 2 steps
SYBR™ Green I Nucleic Acid Gel Stain - 10,000X concentrate in DMSOInvitrogen - Thermo FisherCatalog #S7563
Step 21
IgA Antibody, anti-human, APCMiltenyi BiotecCatalog #130-113-472
Step 9
Normal Mouse SerumJackson ImmunoResearch Laboratories, Inc.Catalog #015-000-120
Step 9
Preparation of fecal samples
Preparation of fecal samples
10m
10m
Weigh out 300 mg of thawed human fecal sample in 2 mL centrifuge tube
Vigorously vortex and mix samples via pipetting to ensure homogenization
Rehydrate sample with 1.25 mL cold 1X PBS (Phosphate-buffered saline ) . Incubate 00:05:00 on ice
5m
Centrifuge samples at 500 x g, 00:15:00
15m
Following centrifugation, filter the clarified supernatant (~1mL) through a 70 μm filter
Remove and retain 100 µL of filtered supernatent for use as the "unsorted" or "native" fraction for subsequent metagenomic sequencing. Keep this fraction on ice for remainder of protocol.
Divide the remaining supernatant (~900 uL) into 2 separate wells of a 96 deep-well plate. These wells will be treated as individual samples throughout the remainder of the protocol, and the two samples will combined at the end of the protocol to increase sample biomass.
Antibody staining
Antibody staining
10m
10m
Prepare the following staining buffer in PBS:
- 3% Fetal Bovine Serum
- 0.5% Sodium Azide
- 1 mM EDTA
Keep staining buffer on ice
Prepare the following antibody master-mix in the staining buffer:
- 1:30 dilution of IgA Antibody, anti-human, APCMiltenyi BiotecCatalog #130-113-472
- 1:50 dilution of Normal Mouse SerumJackson ImmunoResearch Laboratories, Inc.Catalog #015-000-120
Centrifuge the 96 deep-well plate at 5000 x g, 00:05:00
5m
Discard supernatant and resuspend pellets in 100 µL antibody master-mix. Incubate on ice for 00:30:00
30m
Wash samples with 1 mL cold staining buffer. Centrifuge 5000 x g, 00:05:00
5m
Discard supernatent and resuspend pellet in 200 µL staining buffer
Retain 20 µL of the resuspend pellet to act as a stained, unsorted control for Flow Cytometry analysis to establish background to calculate “Native IgA binding percentage”
Magnetic cell separation
Magnetic cell separation
35m
35m
Stain the remaining 180 µL with 20 µL of EasySep APC Selection CocktailEasySep™ APC Positive Selection Kit IISTEMCELL Technologies Inc.Catalog #17681 for 00:15:00 at room tempterature
15m
Add 22 µL of EasySep Dextran RapidSpheres 50100 EasySep™ APC Positive Selection Kit IISTEMCELL Technologies Inc.Catalog #17681 and incubate for 00:10:00 at room temperature
10m
Place samples on
Equipment
MO BIO PowerMag Magnetic Separator
NAME
MO BIO PowerMag Magnetic Separator
BRAND
27400
SKU
for 00:05:00 to allow for magnetic separation.
5m
Carefully discard unbound supernatent while on the magnet. Then remove 96-well plate from magnet and wash with 200 µL of staining buffer.
Repeat steps 17 and 18 an additional 2 times, for a total of 3 00:05:00 magnetic enrichments and washes.
5m
After the final resuspension, combine the duplicate wells (established in Step 7) to establish the "IgA positive" fraction
Flow cytometry
Flow cytometry
Prepare tubes for flow cytometry by combining 5 µL of sample, 20 µL of counting beads, and a 1:10,000 dilution of SYBR™ Green I Nucleic Acid Gel Stain - 10,000X concentrate in DMSOInvitrogen - Thermo FisherCatalog #S7563 into staining buffer for a final volume of200 µL
Tubes should be made using 1) unstained native sample (step 6), 2) stained native sample (from step 14), and 3) stained IgA+ samples (from step 20)
Analyze cells on a flow cytometer
Equipment
FACSymphony™ A5 Cell Analyzer
NAME
BD
BRAND
A5
SKU
Refer to this image to establish flow cytometry gates:
Supplemental Figure S6. Gating strategy for MiG-Seq protocol. The preliminary gate to identify IgA-positive cells was SSC and SYBR Green, with SYBR-positive gate drawn based on preliminary experiments using a sample without SYBR green (SYBR stains all bacteria). SYBR-positive cells were then analyzed for IgA staining, with an IgA-positive gate drawn based on a sample without IgA staining. Representative flow plots are shown for a single sample (#1037) that is unstained (A), staining and unsorted (B), and stained and sorted (C).
For each sample, the native “IgA fraction” is calculated as the number of IgA+ cells in the native sample (b) minus the number of IgA+ cells in the unstained control (a). Samples in which the unstained control (a) hadsa higher number of IgA+ cells than the native stained sample (b), or the native stained sample (b) had a higher number of IgA+ cells than the IgA+ sample (c), should be considered to have “failed QC” and be removed from subsequent analysis.