Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

Summary

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

Abstract

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Introduction

Recent advances in high-throughput sequencing of microbial communities are providing unparalleled sequencing depth and, consequently, an unprecedent glimpse into the functioning and diversity of Earth's microbiome¹. As the ability to multiplex more and more samples onto a single sequencing lane increases, single tube DNA extraction has the potential to become a rate-limiting step in the generation of ecological data. However, new methods in high-throughput DNA extractions hold promise for processing large quantities of environmental samples with greater efficiency than has previously been possible². These methods oft....

Protocol

1. Laboratory bench and tool preparation for loading a 96-well plate

1. Clear bench top by misting with 70% ethanol. Wipe and let air dry before spraying bench top with 10% bleach. Wipe the bench top dry.
2. Sterilize micro-scoopula, spatula, and surgical scissors (curved) by dipping in 95% ethanol and then expose to a flame. Dip each in 10% bleach and allow to air dry prior to use.
   NOTE: Tools should be sterilized between each sample.

2. Sub-sampling and sample preparation

1. Sterilize gloves using ethanol prior to sub-sampling.
2. Homogenize soil samples thoroughly prior to sub-sampling.
....

Results

This novel method was used successfully to load 96-well DNA extraction plates. Comparison of plate loading methods showed our method to have the lowest DNA concentration in the blank wells. The DNA concentration in the blank wells was significantly lower than the method proposed my McPherson et al.⁹ (p < 0.05), though DNA concentrations in our method were not statistically different from the Qiagen default method (Figure 2). All three methods produced mean DNA con.......

Discussion

This method reduces opportunities for well-to-well cross-contamination while loading high-throughput sample extraction plates and offers a more controlled means to load 96-well plates beyond existing plate loading strategies^9,10. Contamination among wells can be more pervasive in 96-well plate extractions than in single-tube extractions, especially when automated^6,7, and, though not specifically tested in.......

Materials

Name	Company	Catalog Number	Comments
18 oz WhirlPak	Nasco	B01065
2 mL centrifuge tubes	Fisherbrand	05-408-129
200 µL micro-PCR tubes	Thermo Scientific	AB 2000
96-well PowerSoil DNA extraction kit	Qiagen	12955-4	We used a soil extraction kit but any 96-well format kit would work.
Ice block for 2 mL centrifuge tubes	Any	Any	Any ice block made for 2 mL tubes will work
Ice block for 200 µL micro-PCR tubes	Any	Any	Any ice block made for 200 µL tubes will work
Micro scoopula	Any	Any
Precut Pierceable Sealing Film	Excel Scientific	XPS25
Spatula	Any	Any
Surgical scissors	Any	Any