Mammalian Genotyping Using Acoustic Droplet Ejection for Enhanced Data Reproducibility, Superior Throughput, and Minimized Cross-Contamination

Cain-Hom, Carol; Pabalate, Ryan; Pham, Anna; Patel, Hetal; Wiler, Rhonda; Cox, Jürgen

doi:10.1177/2211068215601637

Cited by 16 publications

(10 citation statements)

References 35 publications

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“…Other experimental approaches to reduce the impacts of well-to-well contamination bear further investigation. These might include use of higher-fidelity liquid handling approaches [33], or broader adoption of unique-per-sample positive control spike-ins to allow the direct observation and statistical disambiguation of cross-contamination [34]. Methods which rely on identifying and subtracting putative contaminants from datasets need to be used with extreme caution, particularly if the identified sequence variants are present in primary samples.…”

Section: Discussionmentioning

confidence: 99%

Quantifying and understanding well-to-well contamination in microbiome research

Minich

Sanders

Almasi‐Hashiani

et al. 2019

Preprint

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Microbial sequences inferred as belonging to one sample may not have originated from that sample. Such contamination may arise from laboratory or reagent sources or from physical exchange between samples. This study seeks to rigorously assess the behavior of this often-neglected between-sample contamination. Using unique bacteria each assigned a particular well in a plate, we assess the frequency at which sequences from each source appears in other wells. We evaluate the effects of different DNA extraction methods performed in two labs using a consistent plate layout including blanks, low biomass, and high biomass samples. Well-to-well contamination occurred primarily during DNA extraction, and to a lesser extent in library preparation, while barcode leakage was negligible. Labs differed in the levels of contamination. DNA extraction methods differed in their occurrences and levels of well-to-well contamination, with robotic methods having more well-to-well contamination while manual methods having higher background contaminants. Well-to-well contamination was observed to occur primarily in neighboring samples, with rare events up to 10 wells apart. The effect of well-to-well was greatest in samples with lower biomass, and negatively impacted metrics of alpha and beta diversity. Our work emphasizes that sample contamination is a combination of crosstalk from nearby wells and background contaminants. To reduce well-to-well effects, samples should be randomized across plates, and samples of similar biomass processed together. Researchers should evaluate well-to-well contamination in study design and avoid removal of taxa or OTUs appearing in negative controls, as many will be microbes from other samples rather than reagent contaminants.ImportanceMicrobiome research has uncovered magnificent biological and chemical stories across nearly all areas of life science, at times creating controversy when findings reveal fantastic descriptions of microbes living and even thriving in once thought to be sterile environments. Scientists have refuted many of these claims because of contamination, which has led to robust requirements including use of controls for validating accurate portrayals of microbial communities. In this study, we describe a previously undocumented form of contamination, well-to-well contamination and show that contamination primarily occurs during DNA extraction rather than PCR, is highest in plate-based methods as compared to single tube extraction, and occurs in higher frequency in low biomass samples. This finding has profound importance on the field as many current techniques to ‘decontaminate’ a dataset simply relies on an assumption that microbial reads found in blanks are contaminants from ‘outside’ namely the reagents or consumables.

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Section: Discussionmentioning

confidence: 99%

Quantifying and understanding well-to-well contamination in microbiome research

Minich

Sanders

Almasi‐Hashiani

et al. 2019

Preprint

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“…Alpha- and beta-diversity measures were highly correlated across methods. While we did not explicitly test well-to-well contamination, other studies have demonstrated that contamination may be lower when using acoustic liquid handlers over traditional pipette-based liquid handlers for molecular assays while also having higher dispensing accuracy ( 9 ). Finally, while we demonstrate the utility of this method for the 16S rRNA gene, this method could easily be applied to any amplicon based library preparation method.…”

Section: Discussionmentioning

confidence: 99%

High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity

et al. 2018

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Reduced costs of sequencing have tremendously impacted the field of microbial ecology, allowing scientists to design more studies with larger sample sizes that often exceed 10,000 samples. Library preparation costs have not kept pace with sequencing prices, although automated liquid handling robots provide a unique opportunity to bridge this gap while also decreasing human error. Here, we take advantage of an acoustic liquid handling robot to develop a high-throughput miniaturized library preparation method of a highly cited and broadly used 16S rRNA gene amplicon reaction. We evaluate the potential negative effects of reducing the PCR volume along with varying the amount of gDNA going into the reaction. Our optimized method reduces sample-processing costs while continuing to generate a high-quality microbiome readout that is indistinguishable from the original method.

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“…These types of tipless transfers possess the major benefit of eliminating cross-contamination issues. Compared to tip-based dispending, acoustic liquid handling has also been shown to lead to more accurate biological activity in pharmacological assays [33]. This might be due to possible interactions between additives in the plastic tips and reagents, which have been observed to possibly leach from laboratory plasticware [33].…”

Section: Overview Of Technologiesmentioning

confidence: 99%

Unlocking the efficiency of genomics laboratories with robotic liquid-handling

Tegally¹,

San²,

Giandhari³

et al. 2020

BMC Genomics

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In research and clinical genomics laboratories today, sample preparation is the bottleneck of experiments, particularly when it comes to high-throughput next generation sequencing (NGS). More genomics laboratories are now considering liquid-handling automation to make the sequencing workflow more efficient and cost effective. The question remains as to its suitability and return on investment. A number of points need to be carefully considered before introducing robots into biological laboratories. Here, we describe the state-of-the-art technology of both sophisticated and do-it-yourself (DIY) robotic liquid-handlers and provide a practical review of the motivation, implications and requirements of laboratory automation for genome sequencing experiments.

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Mammalian Genotyping Using Acoustic Droplet Ejection for Enhanced Data Reproducibility, Superior Throughput, and Minimized Cross-Contamination

Cited by 16 publications

References 35 publications

Quantifying and understanding well-to-well contamination in microbiome research

Quantifying and understanding well-to-well contamination in microbiome research

High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity

Unlocking the efficiency of genomics laboratories with robotic liquid-handling

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