Not All Quiet on the NGS-Library Front

The results of a scientific procedure usually depend on the quality of the sample, and that is absolutely the case in next-generation sequencing (NGS). Here, preparing a library is a crucial step in sample preparation. The specific steps depend on the sample and the platform. This article reviews a few options.

The key challenges in designing an NGS library focus around the selection and enrichment of the target you want to sequence.

“The key challenges in designing an NGS library focus around the selection and enrichment of the target you want to sequence,” says Emily Leproust, CEO of Twist Bioscience. “It is critically important to know which regions you want to sequence, and then design the library to avoid off-target sequences and avoid areas too close to the areas you’d like to sequence.” While the method must avoid off-target sequences, it must capture all of the targeted sequence. “This requires extreme precision,” Leproust explains.

Pushing up the precision

To increase the odds of effective sequencing, the intended target sequences can be enriched. The sequence, though, impacts enrichment. “Targets with very high or very low GC present a challenge for enrichment protocols that rely on a single temperature for hybridization,” Leproust notes. “High GC targets will require a higher hybridization temperature, while lower GC targets that have hybridized to their complement probe will melt off at higher temperatures.”

The steps in preparing a library require some iterative design adjustments and optimization. To speed up that process, Twist Bioscience provides exome and custom target-enrichment solutions.

“The quality of the sample is a critical determinant of the quality of the resulting library as this process, which relies heavily on enzymatic action, requires the input nucleic acid to be double-stranded and have ends that are amenable to ligation so universal adapters can be added on to enable massively parallel sequencing,” explains Leproust.

When building an NGS library, the use of some samples, such as formalin-fixed paraffin-embedded (FFPE) materials, can be difficult. “Tissue stored in paraffin blocks kept over a number of years at room temperature, which is standard practice, yields DNA that is highly degraded,” Leproust says. “Depending on the isolation method used, the degree of fragmentation and damage could vary greatly leading to very inefficient conversion into a sequenceable library.”

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To produce improved libraries, Twist Bioscience uses double-stranded DNA probes, optimized amplification, and NGS quality control of the probes. These techniques result “in extreme uniformity of the enriched targets with unparalleled specificity,” Leproust explains. “Importantly, we believe the uniformity of our capture means you can effectively run FFPE samples and no longer over sequence your samples—providing future cost savings in terms of increasing the number of samples you can run, increasing the depth of coverage, or reducing overall costs.”

Some technical changes also let scientists use NGS in more places. For example, “the use of molecular barcodes has enabled a variety of high-sensitivity applications, especially in translational and clinical research,” Leproust notes. This arises, in part, from attaching molecular barcodes to fragments. “Tagging these fragments provided a way to increase the sensitivity of detection of certain NGS assays down to sub 1% or to reassemble related pieces of DNA to gain information on paternal or maternal inheritance,” she adds.

Setting up for SMRT sequencing

With SMRT Sequencing from Pacific Biosciences, library preparation includes some specific features. For one thing, this platform works with a “wide range of sample characteristics,” says Paul Kotturi—director of product management, consumables. “These characteristics are typically segmented along the insert size and whether the sample is a fixed length—for example, amplicons—or a range of insert sizes—such as, randomly sheared gDNA.”

To accommodate this range of samples, PacBio developed one workflow for them all. “PacBio recommends slight nuances in the protocol—such as a different concentration of AMPure PB beads—for different insert sizes or sample type for optimal success,” Kotturi notes. “PacBio clearly names our protocols to reflect these different options.”

Although Kotturi points out that he’s seen high success among scientists building libraries for SMRT Sequencing, he believes “the challenges in preparing an NGS library for SMRT Sequencing are related to the qualification of samples with high molecular weight gDNA—that is, greater than 50 kilobases.”

With PacBio’s latest SMRTbell Express Template Prep Kit, though, even libraries composed of long chains of nucleotides are easy. “The kit has a simplified workflow, eliminating the need for AMPure clean-up steps and removing/combining some enzymatic steps during the library process to create an addition-only single tube workflow,” Kotturi explains. “This change minimizes the losses incurred at these steps and decreases the required input amount by 50%.” It’s also fast—cutting preparation from about a day and a half to just more than three hours.

When reaching for RNA

Although NGS can sequence any nucleotide, some technologies aim at RNA. “Each step of NGS library preparation has its own unique challenges,” says Whitney Pike, application scientist at Advanced Analytical Technologies (AATI). “RNA samples should be checked for quality, as they are susceptible to RNases, which can cause degradation and reduces sequencing performance.” To perform that check, Pike recommends electrophoresis.

The Fragment Analyzer from AATI can be used to analyze RNA that will be used to make an NGS library. “By using this platform to analyze the input materials and the nucleic-acid samples during library preparation, problems can be easily identified, and adjustments can be made before they impair the sequencing process,” Pike explains. “When a scientist makes a sequencing library using RNA samples, most times the library can only be used if the sample has a quality score above 7.0, which means the ribosomes are intact with only slight degradation.”

To perform accurate and repeatable NGS, the input nucleotides—DNA or RNA—need to be of the right quality for building a library. Then, following the right steps for the intended sequencing platform must be followed. Only then can scientists build a library worth reading.