As next-generation sequencing (NGS) becomes more routine and experiments are progressively scaling up, library generation can often be a rate-limiting step. Given that library quality is one of the most important determinants of success for NGS, ensuring the highest quality in every aspect of library preparation must be a primary focus. A low-quality library can affect NGS data by skewing results, affecting reliability of reads, and causing issues with reproducibility. Following these guidelines and considering design versatility depending on application, instrument choice, and read length researchers can generate high-quality libraries, creating the foundation for robust experimental results.
Considering the quality of input material
The quality of material going into a library greatly influences the quality of the library and downstream sequencing results. Starting with high-quality and high molecular weight nucleic acids is imperative for achieving long read lengths and precise sequencing performance. High-quality DNA is characterized as predominantly high molecular weight with an A260/280 ratio between 1.8 and 2.0, and free of contaminating substances such as phenol or ethanol.
Starting with high-quality and high molecular weight nucleic acids is imperative for achieving long read lengths and precise sequencing performance.
Ensuring good extraction without causing nucleic acid damage can make library construction a more manageable process. Revvity focuses on developing quality library preparation methods from sample to result. Automated nucleic acid extraction using the chemagic™ bead-based extraction technology delivers high purity and yield from nucleic acid extractions. chemagic technology uses proprietary magnetic particles with a high affinity to nucleic acids and low protein binding, producing ultrapure and high molecular weight nucleic acids.
“The better the extraction chemistry, the better the starting material, and thus the better the NGS results will be,” says Mark Dupal, product manager at Revvity.
For quantitation and quality control, Revvity’s DropletQuant UV-VIS microvolume spectrophotometer provides fast, full-spectrum assessment of sample impurities before moving to downstream processing. Material is analyzed by spectrometry, leveraging the benefits of microfluidics to provide a spectral scan for contaminant analysis. Spectra can be associated with a library of known contaminants, identified contaminants can be removed from the spectra, and an isolated spectral concentration can be read, along with purity analysis.
Revvity is collaborating with 10X Genomics® to further improve the extraction and quantification of various input materials. The 10x Genomics® Chromium™ Genome Solution tags short reads from high molecular weight genomic DNA fragments with molecular barcodes. Combining this with Revvity’s chemagic technology and the LabChip® GX Touch™ Nucleic Acid Analyzer creates an efficient, automated workflow to maximize high molecular weight gDNA extraction, sample QC, and sample prep.
Increasing complexity while decreasing bias
High-quality sequencing libraries should provide even coverage with minimal bias. Ideally, a final NGS library would perfectly represent its starting material so when sequenced, reads are evenly distributed across the entire region of interest. Unfortunately, this is difficult to achieve. There are most likely areas of under-representation with little to no coverage, as well as areas of over-representation with an abundance of coverage. One contributing factor to skewed coverage uniformity is poor library complexity.
“A highly complex library that reflects the original complexity of the source material with high fidelity needs a high-efficiency library prep workflow capable of converting as many input molecules as possible into final library molecules that will be both unique and sequenceable,” explains Jennifer Pavlica, applications manager for the sequencing and life sciences division at Roche.
The KAPA Hyper Prep/Hyper Plus Kits contain a streamlined workflow and are well-known for their high conversion rates and quality. The kits have a highly efficient ligation module to drive conversion of input molecules to final library molecules, and are optimized for various applications with adjustable workflows for sample type and input amount.
With improved complexity comes a reduction in duplication rates, which in turn improves mean coverage and coverage uniformity. Imperfect conformity issues reinforce the importance of limiting bias during library amplification, especially since GC content can play a role in how evenly a target is covered. The KAPA HiFi DNA Polymerase, provided in all Roche library prep kits, helps to minimize GC bias and maintain high complexity.
Roche’s KAPA Hyper Prep Kit can be used to construct libraries for a variety of applications and sample types, including from ultra-low DNA inputs. For example, ChIP-Seq, chromatin immunoprecipitation followed by NGS, is a valuable method for studying DNA interactions with proteins such as transcription factors and histone modifications. The KAPA Hyper Prep Kit with KAPA HiFi HotStart DNA Polymerase enables the examination of low-input and rare samples, resulting in improved sequence coverage and reduced bias. Optimization of library construction parameters is also critical to ensuring successful sequence-quality libraries are generated. By evaluating the ligation efficiency of several DNA input amounts at multiple adapter concentrations the lower limit of the workflow and impact on reproducibility can be determined.
Confidence in sequencing with clean-up and barcoding
Obtaining a clean library is a critical aspect of NGS library generation. Dan Heard, genomics business manager at Qiagen elaborates, “Different clean-up steps such as bead-based or column-based purification are used at different points in the library prep process depending on the method chosen. Until recently, gel excision has been required in the miRNA-Seq workflow for size selection and to reduce adapter-dimers, and is a time-consuming process. Our technology utilizes highly optimized chemistry and successive bead cleanup steps for fast prep and yields very clean libraries.”
Qiagen’s QIAseq 1-Step Amplicon Library Kit builds in two additional clean-up steps to ensure high-quality output from library generation. The one-tube protocol eliminates the need for transferring reagents, increasing efficiency and more effectively capturing insert amplicons, while reducing the risk of contamination. The method can be PCR-free to avoid introducing sequence duplicates or PCR bias, and generates high-quality libraries from a range of input materials.
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Molecular barcoding also helps to instill confidence in quality sequencing runs by tracking original DNA or RNA molecules, calling rare variants, obtaining accurate and quantitative copy number and expression data, and increasing multiplexing ability. Qiagen’s Digital NGS technology allows unbiased quantification of DNA, RNA, and miRNA with NGS, leveraging Qiagen’s Molecular Index Technology to enable more accurate quantification and detection of molecular variants across all sequencing platforms.
Christopher Mason, associate professor at Weill Cornell Medicine, collaborated with Qiagen to perform a metagenomic analysis of the New York City subway system. The study, called PathoMap, took a comprehensive look at microbes around the city to gain a baseline understanding of what humans come into contact with on a daily basis. Qiagen’s sample collection, extraction, and library preparation methods aided in creating high-quality libraries for NGS analysis, helping the team discover novel DNA from unidentified species and successfully characterize a large assortment of microbes. PathoMap has also led to an international consortium that is replicating the study in over 70 cities, in a project called the metagenomics and metadesign of subways and urban biomes (MetaSUB).
New methods in single-cell sequencing
Single-cell sequencing is a challenging approach that investigates the genomes or transcriptomes of individual cells, providing an in-depth assessment of cell-to-cell variation. Manipulating individual cells is much more difficult than examining large populations, and with only a tiny amount of RNA coming from each cell, there is no room for error. Illumina’s SureCell Whole Transcriptome Analysis (WTA) 3’ Kit provides a validated process for single-cell RNA sequencing, allowing generation of high-quality product from low quantity samples.
Illumina product manager, Kevin Taylor, explains, “The SureCell™ WTA 3’ Library Prep Kit is the first kit launched as part of the Illumina Bio-Rad Single Cell Sequencing Solution. What makes it unique relative to other droplet-based single-cell solutions is that the single-cell libraries are generated using direct cDNA tagmentation using Nextera technology without the need for shearing or pre-amplification.”
RNA sequencing (RNA-Seq) methods support the exploration of individual contributions of single cells in tissues of interest. A high-quality library enables ultra-low volume input and single-cell RNA-Seq to supplement the study of cell function with a detailed evaluation of heterogeneity in differentiation, proliferation, and tumorigenesis processes.
Seeing into the future
Given that NGS is so prevalent in genomics research, consistent process improvements will continue to provide higher quality products from sample extraction, purification, library generation, and downstream processing. Speed and simplicity of quality library preparation is a focus for companies, working to create procedures that ensure sequencing success regardless of input. Companies like Revvity are taking improvements one step further with artificial intelligence. Revvity’s IntelliChip™ technology minimizes standard deviation and variance from run to run for extreme assay optimization, providing intra- and inter-run reproducibility. Innovations such as this will continue to bring NGS into the future for further investigation of our living world.
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