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Introduction

Next-generation sequencing (NGS) has revolutionized the field of genomics, enabling researchers to study vast amounts of genetic information quickly and cost-effectively.

However, even the slightest errors in sample and library preparation can have significant consequences on the accuracy and reliability of results. For example, a pipetting inaccuracy of as little as 5% can result in a 2 ng variation in template DNA.1

In this article, we will explore common errors that occur during the sample handling and library preparation phases and provide evidence on how to mitigate these concerns.

Errors in NGS Sample Preparation

NGS labs are being asked to handle more challenging samples like those with diverse origins, lower quality, or smaller size.2 So sample preparation is increasingly crucial to obtaining reliable results.

One consideration is inaccurate handling of samples, which can lead to cross-contamination and degradation between samples.3 To prevent this, it is essential to thoroughly sterilize your workstation and tools prior to the sample preparation process. Additionally, consider handling one sample at a time, which minimizes the risk of unintentional mixing.

As a quality control (QC) measure, include DNA-free samples alongside your actual samples. This helps identify any potential contamination during this step in the process. Further, it is vital to regularly monitor and document QC measures to ensure the integrity of your results.

Proper PCR amplification and sequencing read depth rely on the use of an adequate amount of high-quality DNA. For most applications, it is recommended to have a minimum of 200-500 ng of total DNA template. Using less than the required input can increase risk of error and lead to low sequencing coverage and unreliable data.

Errors in NGS Library Preparation

Library preparation plays a crucial role in NGS, and errors at this stage can significantly impact the accuracy and quality of the final data.

For example, biases in primer binding or “mispriming” during the library prep phase can result in uneven coverage of the target regions. To minimize these errors, carefully design primers and ensure they are specific to your intended targets. Optimizing PCR conditions and using high-quality primers can also reduce the risk of mispriming.4

Finally, when processing multiple samples simultaneously, batch effects can arise due to variations in reagents, equipment, or operator-related factors.5 Batch effects can greatly affect the outcome of gene expression analysis, and that effect is much more severe for miRNA-seq data.6 To minimize batch effects, you can randomize sample processing across different batches and include positive controls for each batch.

Multiplexing to Mitigate Error

Sample multiplexing is necessary for meeting demands in high-throughput settings. Multiplexing also makes economical use of available sequencing capacities. However, scaling up a workflow is not without its challenges, such as:7

  • Index misassignment
  • Inconsistencies with read depth

The ExpressPlex™ Library Prep Kit mitigates these concerns by offering simplicity while maintaining high-throughput, multiplexed, and scalable sample and library preparation. This kit requires just 30 minutes of hands-on time to sequence 96 samples.

“ExpressPlex combined three or four steps into one, which is unique to seqWell products. That made our library prep so much faster, and the quality control was significantly better compared to other kits. Now the risk of error has decreased significantly for our technicians.”

– Lacy Simons, lab director of the Center for Pathogen Genomics and Microbial Evolution at Northwestern University

 

Further, ExpressPlex features significantly higher levels of auto-normalization compared to competitors. Users can maintain read counts and insert sizes across a 10-fold range. So individual normalization is no longer required to achieve more consistent read depths across samples.

ExpressPlex is also easily automated, which circumvents the errors often associated with manual pipetting. This protocol was designed to support automation and flexible reaction volumes. It requires just two pipetting steps per sample prior to placing the full plate onto the thermocycler and users can maximize efficiency with a robotic liquid handling platform. Click here to review data demonstrating the effectiveness of ExpressPlex in combination with SPT Labtech’s firefly® liquid handler, and click here to review data on ExpressPlex in combination with the Tecan Fluent® liquid handler to create compact and cost-efficient methods for fast screening of synthetic constructs.9,10 

The team at Octant Bio leveraged the speed and simplicity of ExpressPlex in the development of their high-throughput plasmid sequencing pipeline update. Click here to watch our joint webinar with Octant where we discuss this collaboration.8

Conclusion

Accurate sample and library preparation are essential to obtaining reliable and high-quality NGS data. Investing time and attention into robust sample and library preparation will undoubtedly pay off in more accurate and meaningful genomic insights.

By following best practices, such as maintaining a sterile environment, using adequate DNA amounts, and addressing common library preparation challenges, researchers can minimize errors and ensure the integrity of their sequencing results.

Further, labs can leverage the speed and simplicity of ExpressPlex to easily train their team, avoid costly errors, and spend more time focusing on results. Click here to watch the ExpressPlex protocol demonstration video and see how easy this kit is to use.11 We also offer in-person ExpressPlex demos so get in touch with us if you’re interested.

References

  1. BioSizeBio | Pipetting inaccuracies
  2. Front Line Genomics | Sample prep for NGS
  3. Cheeky Scientist | The do’s and don’ts of NGS
  4. Genome Research | Reduction of mispriming in amplification reactions with restricted PCR
  5. BMC Bioinformatics | Identifying and mitigating batch effects in whole genome sequencing data
  6. Translational Cancer Research | Statistical strategies for microRNAseq batch effect reduction
  7. Technology Networks Genomics Research | Tackling the challenges of multiplexing in NGS
  8. seqWell | Enabling High-Throughput Biology with New Technologies for Synthetic Construct Sequencing
  9. seqWell | High-Throughput NGS Library Prep for Plasmids and Amplicons with the ExpressPlex™ Library Preparation Kit on the SPT Labtech firefly
  10. seqWell | High-Throughput NGS Library Prep for Plasmids and Amplicons Using the ExpressPlex™ Library Preparation Kit on the Tecan Fluent®
  11. seqWell | ExpressPlex™ Library Prep Kit Single-Step Workflow Demo for High-Throughput Multiplexed Sample Preparation