TITLE: Nucleic Acid Purification: Bead Clean
AUTHORS: Madeline Mayday, Lillian Khan, Eric D. Chow, Matt S. Zinter, Joseph L. DeRisi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS).  While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process.  Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages.  Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries.  For 384-well mNGS library preparations, we achieved a savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">This automated, miniaturized protocol for bead cleaning can be used with the High-Throughput Echo Library Prep Protocol for processing up to 384 samples simultaneously.  To facilitate the miniaturized columes required by this protocol, we have included the .STL file for our 3D-printed adaptor, which, when placed on the magnet, raises the height of the PCR plate, thereby lowering the beads inside of the well and allowing for elutions in as low as 6uL.</div></div></div>

[STEPS]
?. [Add Beads]
Aspirate desired volume of Ampure beads from reservoir and mix well into sample plate by mixing total volume minus  one microliter less -- 20 times.
Bead volumes for each bead clean are available in the High-Throughput Echo Library Prep Protocol.
?. [Incubate]
Transfer plate onto Alpaqua 384 Post Magnet Plate with 3D-printed adaptor and incubate at room temperature for another 5 minutes to allow for the formation of a magnetic pellet.
It is imperative that the magnet used can elute, at the very minimum, 6uL of your chosen elution buffer. It is important to elute cDNA in as little volume as possible for a miniaturized protocol.
?. [Air dry pellet]
Remove and discard the supernatent for the second wash step. Allow the magnetic pellet to air dry while on the magnetic rack for a maximum of 90 seconds.
Do not overdry the beads. This could cause undesired loss of cDNA.
?. [Ethanol washes]
Aspirate 25uL freshly prepared 80% ethanol from reservoir and add to samples while on the magnetic rack.  Incubate at room temperature for 30 seconds.
?. [Resuspend]
Remove plate from magnetic stand, and resuspend beads in desired elution buffer and volume. Mix well.
Volume and type of eluent are specified in the High-Throughput Echo Library Prep Protocol.Reminder: Regular elution buffer is used for Post-Second Strand Synthesis, Post- End Prep, and Post- USER/PCR bead cleans. Barcodes suspended in nuclease-free water will be used to elute in Post-Adaptor Ligation bead clean.
?. [Incubate]
Incubate at room temperature for 5 minutes.  Transfer plate onto Alpaqua 384 Post Magnet Plate with adaptor with 3D-printed adaptor and incubate at room temperature for another 5 minutes.
?. [Recovery of Purified cDNA]
Slowly aspirate supernatant, now containing cDNA, and transfer to final PCR plate.
?. [Prep Reagents]
Prepare all necessary reagents and bring to room temperature:  AB1ReagentCatalog Number2AMPure XP beadsA63881380% Ethanol4Desired Elution Buffer (H2O, elution buffer, etc)5Alpaqua 384 well Post Magnetic PlateSKU A0012226Low elution adaptor
AB1ReagentCatalog Number2AMPure XP beadsA63881380% Ethanol4Desired Elution Buffer (H2O, elution buffer, etc)5Alpaqua 384 well Post Magnetic PlateSKU A0012226Low elution adaptor
?. [Incubate]
Incubate samples and beads at room temperature for 5 minutes.
?. [Ethanol washes]
Slowly aspirate supernatant and discard.  Repeat the wash step for a total of 2 washes.