Nov 02, 2020
Version 1
Preparing biological samples for metabarcoding V.1
- 1The Roslin Institute, University of Edinburgh
Protocol Citation: Tim Regan 2020. Preparing biological samples for metabarcoding. protocols.io https://dx.doi.org/10.17504/protocols.io.bkyrkxv6
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: In development
We are still developing and optimizing this protocol
Created: September 07, 2020
Last Modified: November 02, 2020
Protocol Integer ID: 41713
Keywords: Metabarcoding, metagenomics, DNA extraction,
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Abstract
This protocol describes the preparation of biological samples (specifically from a marine environment e.g. hatchery or RAS unit) for amplicon sequencing. Starting with a biological sample stored in Qiagen buffer ATL, or similar, it begins with a bead beating process to homogenise the sample. Enzymatic lysis using Metapolyzyme and Proteinase K are emplyed to ensure efficient DNA release. The Qiagen DNeasy kit is used to column extract DNA from lysates. Following concentration estimates of DNA elutions, samples are diluted >1:10 to avoid PCR inhibition during amplicon library preparation.
Guidelines
In every step following enzymatic digestion of samples (and in general), ensure samples are kept at 4C to maximise sample stability.
Freeze DNA samples if not being used for >1 week following extraction.
Otherwise, storing DNA at 4C in fridge is preferable.
Materials
MATERIALS
Buffer ALCatalog #19075
QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
Buffer ATLQiagenCatalog #19076
Proteinase K, 100mgPromegaCatalog #V3021
PBS
Ethanol 70%
MetaPolyzymeSigma AldrichCatalog #MAC4L-5MG
UltraPure™ DNase/RNase-Free Distilled WaterThermo FisherCatalog #10977015
Lysing Matrix A 2 mL tubeMP BiomedicalsCatalog #SKU 116910050-CF
Centrifuge.
Bead beater.
Incubator (for 37C and 56C).
Pipettes and tips.
Protocol materials
Proteinase K, 100mgPromegaCatalog #V3021
UltraPure™ DNase/RNase-Free Distilled WaterThermo FisherCatalog #10977015
Lysing Matrix A 2 mL tubeMP BiomedicalsCatalog #SKU 116910050-CF
Buffer ALCatalog #19075
QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
Ethanol 70%
MetaPolyzymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #MAC4L-5MG
Buffer ATLQiagenCatalog #19076
PBS
Buffer ATL QiagenCatalog #19076
Lysing Matrix A 2 mL tubeMP BiomedicalsCatalog #SKU 116910050-CF
MetaPolyzymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #MAC4L-5MG
Proteinase K, 100mgPromegaCatalog #V3021
Buffer AL, Lysis bufferQiagenCatalog #19076
QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
Safety warnings
Refer to manufacturer's MSDS information for each reagent used to ensure appropriate and safe use.
Before start
Ensure leaving time for samples to thaw if frozen. Avoid leaving samples thaw for too long as this may lead to degradation.
Bead beating
Bead beating
45m
45m
Starting with biological sample (filter, swab, water, biofilm, tissue etc.) stored in Qiagen Buffer ATL (or similar), transfer up to 1 mL to Matrix A bead tube.
Buffer ATL QiagenCatalog #19076
Lysing Matrix A 2 mL tubeMP BiomedicalsCatalog #SKU 116910050-CF
30m
Perform bead beating in a disruptor at at 5.0 M/s (speed) for 00:00:40 x2 (ensure tube looks homogenous).
15m
Enzymatic digestion
Enzymatic digestion
2h 15m
2h 15m
Add 5 µL of Metapolyzyme to each tube and vortex briefly.
Incubate samples at 37 °C for 02:00:00 .
MetaPolyzymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #MAC4L-5MG
2h 15m
Add 20 µL of Proteinase K to each tube, vortex for 00:00:10 , then incubate at 56 °C Overnight .
Proteinase K, 100mgPromegaCatalog #V3021
16h
DNA extraction
DNA extraction
Vortex samples for 00:00:15 and centrifuged 13000 x g for 00:01:00 .
Transfer the supernatant from each tube (up to 900 µL ) into a new tube and centrifuged at 13000 x g, 00:01:00 .
Transfer up to 600 µL of bead-free supernatant to a new 2 mL tube.
Premix 70% ethanol and Qiagen lysis buffer AL 1:1 to add to sample at a ratio of 1:1:1
e.g. for 10 samples of 500 µL each, premix 550 µL of buffer AL and 550 µL of 70% ethanol and add 1 mL of ethanol/buffer AL mixture to each sample.
Buffer AL, Lysis bufferQiagenCatalog #19076
Hereafter, the manufacturer's protocol for the Qiagen DNeasy Blood and Tissue kit is followed with some modifications:
- Load <600 µL of lysate mixture (ATL, AL and EtOH) at a time into the column
- Spin at 6000 x g, 00:01:00 and discard flow-through.
- Repeat as necessary until all lysate is loaded on column e.g. mixture of 1500 µL may take x3 initial spins and flow through discarding to complete column binding.
QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
- Place the DNeasy Mini spin column in a new 2 mL collection tube (provided), add 500 µL Buffer AW1, and centrifuge a 6000 x g, 00:01:00 (8000 rpm).
- Discard flow-through and collection tube.
- Place the DNeasy Mini spin column in a new 2 mL collection tube (provided), add 500 µL Buffer AW2, and centrifuge for at 20000 x g, 00:03:00 to dry the DNeasy membrane.
- Discard flow-through and collection tube.
Perform final elution in 100 µL of AE buffer.
Preparing concentration for library preparation
Preparing concentration for library preparation
Check approximate concentration of extracted DNA using a Nanodrop.
Prepare 1:10 dilution of each extraction for PCR (to avoid PCR inhibition).
Perform further dilution of sample to a maximum final concentration of ~1 ng/μl - 10 ng/μl
Use ~50 ng of DNA in a 20 µL per sequencing library PCR reaction (see amplicon library PCR protocol).