With so many new technologies on the forefront of genomics and next-generation sequencing (NGS), research is incorporating a wider variety of samples that couldn’t be analyzed with previous product lines. Samples such as those from FFPE tissues, biofluids, tough-to-lyse and inhibitor rich samples for microbiomics, or other samples with little to variable nucleic acid input create a shaky foundation to build from. Using highly sensitive quality control measures and improved extraction and purification techniques, aged, degraded, and contaminated samples can be successfully sequenced.
Quality control
Due to variability in processes for sample collection and storage, quality control becomes one of the most important steps when prepping for NGS. Knowing the level of degradation or chemical modification a sample has gone through can help determine what path you take to rehabilitate a sample or seek a different approach.
Quality control becomes one of the most important steps when prepping for NGS.
Using quality control tools such as Agilent’s 2100 Bioanalyzer or 4200 TapeStation systems to obtain information on nucleic acid quantification, integrity, and sizing of fragments is a good start to ensuring uniformity of fragments for sequencing. Both systems are well-accepted standards, as well as fast and easy automated QC tools that have been included with various NGS workflows. Analyzing nucleic acid purity using 260/280 ratios can ensure samples are clean and clear of contaminants that could inhibit downstream reactions.
The level of degradation is also important to examine, setting a threshold for whether a sample can be sequenced or not. Illumina suggests their TruSeq FFPE DNA Library Prep QC Kit to evaluate sample quality and determine sample viability by a real-time PCR assay. For RNA, using the DV200 index can help determine the amount of FFPE RNA to input in a library prep kit, or simply as a way to decide whether the sample is too degraded to move forward.
Formalin-fixed paraffin-embedded samples
With growing interest in sequencing FFPE samples that have been collected globally for cancer research, investigation of rare diseases, and other applications, companies have focused specifically on solutions to obtain detailed data from these typically degraded and challenging samples.
Common issues seen with FFPE samples include deamination, cross-linking, and degradation, from the process of sample collection and fixation, sample storage inconsistencies with temperature and light, or extraction of the nucleic acid itself. “Every FFPE block can be considered as unique as the tissue it contains. Tumor content, tumor stage, tissue type, can all have a dramatic impact on the ability to isolate nucleic acids, creating real challenges when developing extraction processes. Any single chemistry will be dealing with a widely variable input or starting point,” explains Jung Doh, senior applications scientist at Beckman Coulter Life Sciences.
Beckman is working to address this variability through the entire workflow, from nucleic acid extraction from paraffin using FormaPure optimized to deliver high-quality DNA and RNA for creating sequencing ready libraries to automation of FFPE-optimized NGS library construction kits from multiple vendors on the Biomek Liquid Handling platforms, including NEBNext Ultra II DNA Kit, Swift Biosciences Accel- NGS 2S Plus DNA Library Kit, and Kapa HyperPlus Kit.
Beckman Biomek Automated NGS methods are also commonly partnered with Illumina kits. The Illumina TruSeq Custom Amplicon Low Input kit is an ideal fit for FFPE samples. The kit uses Illumina’s amplicon chemistry by incorporating a dual probe design to help generate data from lesser quality samples and eliminate any false positives that can arise from deamination events during formalin fixation. For RNA, Illumina’s TruSeq RNA Access for FFPE samples allows for less input volume, is optimized for sequencing from degraded samples, and also works with automated systems.
Biofluids and the microbiome
Biofluids and samples intended for microbiomics present challenges such as contamination, presence of inhibitors, and profile alterations associated with sample collection and nucleic acid isolation.
“To give circulating cell-free DNA (ccfDNA) samples the best chance at successful NGS sequencing,” says Angela Ryan, senior director, head of global life sciences strategic marketing at Qiagen, “the QIAseq cfDNA Library Kits, with Qiagens’s Ultralow Input ligation chemistry, allow as much DNA as possible from dilute samples to be converted to either Illumina or Ion Torrent-compatible whole genome libraries.”
To ensure that pre-analytical steps do not interfere with downstream analysis, the PAXgene Blood ccfDNA Tubes and Kits enable the stabilization of a blood sample’s ccfDNA profile, and offer an automated solution for the QIAsymphony instrument for standardized processing and high reproducibility. Qiagen also recently released direct processing of blood collection tubes on the instrument to avoid risk of sample mix up and limit biohazardous waste.
Extracting ccfDNA from plasma or serum can be performed using Qiagen’s new QIAamp MinElute ccfDNA kits. The kits offer high concentration and yield of purified ccfDNA even with low elution volume (20 µl) and scalable input volumes of 1–10 ml. Newly launched automated options are also available with the EZ1 ccfDNA kits that enable streamlined ccfDNA extraction on the EZ1 Advanced XL instrument.
Zymo Research offers a standardized portfolio of products to overcome the challenges in microbiomics. “For microbiomics, the whole community profile and relative abundance of each organism is important, therefore extra care must be taken to ensure that a sample’s profile is preserved because it can change rapidly once it is moved outside of its current environment, creating variable results and findings,” explains Ryan Kemp, director of nucleic acid solutions at Zymo Research.
Kemp notes the importance of freezing a sample immediately upon collection to avoid organism growth or decay and nucleic acid degradation that can severely alter its profile. His suggested sample storage solution is Zymo’s DNA/RNA Shield, a nucleic acid preservation solution to protect nucleic acids and a microbial inactivation solution to prevent new growth combined.
Zymo Research has also tackled the issue of community profile misrepresentation associated with biased lysis of microbes by developing a kit designed specifically for microbiomics. The ZymoBIOMICS DNA Mini kit uses high-density ceramic beads of mixed sizes to ensure unbiased lysis of microbes within a sample.
Low nucleic acid input
Lower input volumes equate to less data. To combat this challenge, companies such as GE have introduced specialty kits for low volume inputs to maximize data output. Their Single Cell Genomiphi kit is specially formulated to provide reliable amplification of representative genomic DNA from single cells for use in NGS library construction. Combining GE’s Nucleon and Genomiphi technologies allows isolation of high-quality genomic DNA molecules present in the starting material in about an hour.
“Researchers need to be able to amplify the entire genome consistently from miniscule amounts, such that they have the best chance of completing as many assays as possible from whatever DNA they are able to isolate from their starting material,” explains Jared Stephens, business development in genomics and cellular research at GE Healthcare Life Sciences.
New England BioLabs has been focusing new technology development in this space as well, introducing the NEBNext® Ultra™ II DNA Library Prep Kit and NEBNext Ultra II RNA Library Prep Kits to meet the challenge of constructing high-quality libraries from ever-decreasing input quantities. The reagents for each step in the library preparation workflow have been reformulated to enable high-yield preparation of high-quality libraries from 500 picograms to 1 microgram of input DNA or with limited amounts of RNA (10 ng–1 μg total RNA with the (poly(A) mRNA workflow or 5 ng–1 μg total RNA with the rRNA depletion workflow). These kits minimize bias with fewer PCR cycles, are automation compatible, and can be used with low-quality RNA, including FFPE.
Future focus
In addition to releasing new technologies and reagents for current issues working with challenging sample types, companies are also looking forward to work out what future challenges researchers will face.
The lack of standardization across sample collection protocols, nucleic acid isolation, and library preparation present a significant challenge. The need for standardized workflows to improve accuracy and reproducibility has been recognized by researchers, commercial organizations, and government organizations including NIST (National Institute of Standards and Technology). In order to standardize and validate such methods, mock microbial communities such as the ZymoBiomics Microbial Community DNA Standard from Zymo Research that include errors/bias measurements and method optimization, validation, standardization, and routine quality control for each experiment are needed.
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“The need for standardized methods and workflows to avoid unreliable or even false analytical test results, including sequencing bias still exists,” explains Qiagen’s Ryan, “Consortia like CANCER-ID help develop standards to enable broad adoption of reliable molecular analyses from liquid biopsies. Qiagen is happy to have joined CANCER-ID to share our experience and contribute with a wide range of solutions.”
With so many different challenges to solve and low input volumes becoming more prevalent with diverse sample types, long-term solutions such as broad range kits are becoming a substantial focus for development. According to Fiona Stewart, product portfolio manager for next generation sequencing at New England Biolabs, “Too many specialized kits create inventory management issues and too many workflows within one lab to organize efficiently and reliably. NEB is working to solve these issues with fewer kits that can do more.”
GE Healthcare Life Sciences offers solutions to nucleic acid length. “Generally, in the context of NGS, length has begun to matter again. New assays are coming online that depend on the isolation of the longest genomic DNA molecules present in the starting material. Products like Nucleon are positioned to enable a growing era of long read, linked read and long range information pursuit by the field,” says Stephens.
Images: Beckman Coulter & Zymo Research