TITLE: Preparing biological samples for metabarcoding
AUTHORS: Tim Regan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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. </div></div>

[STEPS]
?. [Bead beating]
Starting with biological sample (filter, swab, water, biofilm, tissue etc.) stored in Qiagen Buffer ATL (or similar), transfer up to  to Matrix A bead tube.
1 mL
?. [Bead beating]
Perform bead beating in a disruptor at at 5.0 M/s (speed) for  x2 (ensure tube looks homogenous).
?. [Enzymatic digestion]
Add  of Metapolyzyme to each tube and vortex briefly.Incubate samples at   for .
5 µl
37 °C
?. [Enzymatic digestion]
Add  of Proteinase K to each tube, vortex for , then incubate at  .
20 µl
56 °C
?. [DNA extraction]
Vortex samples for  and centrifuged  for .
Centrifuge: 13000 34
?. [DNA extraction]
Transfer the supernatant from each tube (up to ) into a new tube and centrifuged at .
900 µl
Centrifuge: 13000 34, 1 min
?. [DNA extraction]
Transfer up to of bead-free supernatant to a new tube.
600 µl
2 mL
?. [DNA extraction]
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  each, premix of buffer AL and  of 70% ethanol and add  of ethanol/buffer AL mixture to each sample.
500 µl
550 µl
550 µl
1 mL
?. [DNA extraction]
Hereafter, the manufacturer's protocol for the Qiagen DNeasy Blood and Tissue kit is followed with some modifications:Load  of lysate mixture (ATL, AL and EtOH) at a time into the column Spin at  and discard flow-through. Repeat as necessary until all lysate is loaded on column e.g. mixture of  may take x3 initial spins and flow through discarding to complete column binding.
600 µl
Centrifuge: 6000 34, 1 min
1500 µl
?. [DNA extraction]
Place the DNeasy Mini spin column in a new  collection tube (provided), add  Buffer AW1, and centrifuge a  (8000 rpm).
2 mL
500 µl
Centrifuge: 6000 34, 1 min
?. [DNA extraction]
Place the DNeasy Mini spin column in a new collection tube (provided), add  Buffer AW2, and centrifuge for  at  to dry the DNeasy membrane.
2 mL
500 µl
Centrifuge: 20000 34, 3 min
?. [DNA extraction]
Perform final elution in of AE buffer.
100 µl
?. [Preparing concentration for library preparation]
Check approximate concentration of extracted DNA using a Nanodrop.
?. [Preparing concentration for library preparation]
Prepare 1:10 dilution of each extraction for PCR (to avoid PCR inhibition).Perform further dilution of sample to a maximum final concentration of ~ -
?. [Preparing concentration for library preparation]
Use ~ of DNA in a  per sequencing library PCR reaction (see amplicon library PCR protocol).
50 ng
20 µl