Characterization and mitigation of artifacts derived from NGS library preparation due to structure-specific sequences in the human genome.

BACKGROUND: Hybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors. RESULTS: We used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation "blacklist" in the BED region to reduce errors in downstream analyses. CONCLUSIONS: We first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.

Optimization of enzymatic fragmentation is crucial to maximize genome coverage: a comparison of library preparation methods for Illumina sequencing.

BACKGROUND: Novel commercial kits for whole genome library preparation for next-generation sequencing on Illumina platforms promise shorter workflows, lower inputs and cost savings. Time savings are achieved by employing enzymatic DNA fragmentation and by combining end-repair and tailing reactions. Fewer cleanup steps also allow greater DNA input flexibility (1 ng-1 µg), PCR-free options from 100 ng DNA, and lower price as compared to the well-established sonication and tagmentation-based DNA library preparation kits. RESULTS: We compared the performance of four enzymatic fragmentation-based DNA library preparation kits (from New England Biolabs, Roche, Swift Biosciences and Quantabio) to a tagmentation-based kit (Illumina) using low input DNA amounts (10 ng) and PCR-free reactions with 100 ng DNA. With four technical replicates of each input amount and kit, we compared the kits' fragmentation sequence-bias as well as performance parameters such as sequence coverage and the clinically relevant detection of single nucleotide and indel variants. While all kits produced high quality sequence data and demonstrated similar performance, several enzymatic fragmentation methods produced library insert sizes which deviated from those intended. Libraries with longer insert lengths performed better in terms of coverage, SNV and indel detection. Lower performance of shorter-insert libraries could be explained by loss of sequence coverage to overlapping paired-end reads, exacerbated by the preferential sequencing of shorter fragments on Illumina sequencers. We also observed that libraries prepared with minimal or no PCR performed best with regard to indel detection. CONCLUSIONS: The enzymatic fragmentation-based DNA library preparation kits from NEB, Roche, Swift and Quantabio are good alternatives to the tagmentation based Nextera DNA flex kit from Illumina, offering reproducible results using flexible DNA inputs, quick workflows and lower prices. Libraries with insert DNA fragments longer than the cumulative sum of both read lengths avoid read overlap, thus produce more informative data that leads to strongly improved genome coverage and consequently also increased sensitivity and precision of SNP and indel detection. In order to best utilize such enzymatic fragmentation reagents, researchers should be prepared to invest time to optimize fragmentation conditions for their particular samples.

A High-Performance Fluorescence Immunoassay Based on the Relaxation of Quenching, Exemplified by Detection of Cardiac Troponin I.

The intramolecular fluorescence self-quenching phenomenon is a major drawback in developing high-performance fluorometric biosensors which use common fluorophores as signal generators. We propose two strategies involving liberation of the fluorescent molecules by means of enzymatic fragmentation of protein or dehybridization of double-stranded DNA. In the former, bovine serum albumin (BSA) was coupled with the fluorescent BODIPY dye (Red BSA), and then immobilized on a solid surface. When the insolubilized Red BSA was treated with proteinase K (10 units/mL) for 30 min, the fluorescent signal was significantly increased (3.5-fold) compared to the untreated control. In the second case, fluorophore-tagged DNA probes were linked to gold nanoparticles by hybridization with capture DNA strands densely immobilized on the surface. The quenched fluorescence signal was recovered (3.7-fold) by thermal dehybridization, which was induced with light of a specific wavelength (e.g., 530 nm) for less than 1 min. We next applied the Red BSA self-quenching relaxation technique employing enzymatic fragmentation to a high-performance immunoassay of cardiac troponin I (cTnI) in a microtiter plate format. The detection limit was 0.19 ng/mL cTnI, and the fluorescent signal was enhanced approximately 4.1-fold compared with the conventional method of direct measurement of the fluorescent signal from a non-fragmented fluorophore-labeled antibody.

Impact of various factors on the kinetics of non-enzymatic fragmentation of a monoclonal antibody.

Non-enzymatic hinge fragmentation of monoclonal antibodies (mAb) is considered a critical quality attribute since it changes the primary sequence of the proteins, thereby leading to structural changes which can affect stability, function, and efficacy. While peptide bonds are exceptionally stable under physiological conditions, reactive side chains of a few residues, the flexibility of the backbone, and physicochemical parameters such as pH, temperature, and the reaction of radicals and metal ions can promote the cleavage of peptide bonds. In this study, the relative extent and rate of fragmentation are compared with respect to the presence of several different factors (including hydrogen peroxide, metal ion, and temperature) as measured by size exclusion chromatography. A kinetic model of monomer degradation as a function of time (mAb only) is created. In the presence of either H2O2 or Cu2+, or both, the reaction kinetics follow different orders depending on the reaction conditions. The half-life for peptide bond cleavage of the mAb hinge region was 385 days at 40 °C and decreases to 250, 48, and 45 days in the presence of H2O2, Cu2+, and a combination of H2O2 and Cu2+, respectively. A temperature dependence of peptide bond cleavage at 35 °C, 40 °C, 45 °C, and 50 °C showed Arrhenius behavior with an apparent activation energy of 76.9 ± 16.4 kJ/mol. The reaction rates obtained from the Arrhenius equation were then extrapolated to predict fragmentation rates under real storage conditions (e.g., at 2-8 °C). We demonstrate that trace levels of impurities including peroxide left after surface sterilization or degradation of non-ionic surfactants or metal ions from the buffer components can significantly affect the stability of a mAb.

Structural Characterization of Pectic Polysaccharides in the Cell Wall of Stevens Variety Cranberry Using Highly Specific Pectin-Hydrolyzing Enzymes.

The potential of poly- and oligosaccharides as functional ingredients depends on the type and glycosidic linkages of their monosaccharide residues, which determine their techno-functional properties, their digestibility and their fermentability. To isolate the pectic polysaccharides of cranberry, alcohol insoluble solids were first obtained from pomace. A sequential extraction with hot phosphate buffer, chelating agents (CH), diluted (DA) and concentrated sodium hydroxide was then carried out. Pectic polysaccharides present in CH and DA extracts were purified by anion exchange and gel filtration chromatography, then sequentially exposed to commercially available pectin-degrading enzymes (endo-polygalacturonase, pectin lyase and endo-arabinanase/endo-galactanase/both). The composition and linkages of the generated fragments revealed important characteristic features, including the presence of homogalacturonan with varied methyl esterification extent, branched type I arabinogalactan and pectic galactan. The presence of arabinan with galactose branches was suggested upon the analysis of the fragments by LC-MS.

Structural features conserved in subclass of type II arabinogalactan.

Arabinogalactan-proteins (AGPs) are extracellular proteoglycans, which are presumed to participate in the regulation of cell shape, thus contributing to the excellent mechanical properties of plants. AGPs consist of a hydroxyproline-rich core-protein and large arabinogalactan (AG) sugar chains, called type II AGs. These AGs have a ß-1,3-galactan backbone and ß-1,6-galactan side chains, to which other sugars are attached. The structure of type II AG differs depending on source plant, tissue, and age. Type II AGs obtained from woody plants in large quantity as represented by gum arabic and larch AG, here designated gum arabic-subclass, have a ß-1,3;1,6-galactan structure in which the ß-1,3-galactan backbone is highly substituted with short ß-1,6-galactan side chains. On the other hand, it is unclear whether type II AGs found as the glycan part of AGPs from herbaceous plants, here designated AGP-subclass, also have conserved ß-1,3:1,6-galactan structural features. In the present study we explore similarities of type II AG structures in the AGP-subclass. Type II AGs in fractions obtained from spinach, broccoli, bok choy, komatsuna, and cucumber were hydrolyzed into galactose and ß-1,6-galactooligosaccharides by specific enzymes. Based on the proportion of these sugars, the substitution ratio of the ß-1,3-galactan backbone was calculated as 46-58% in the five vegetables, which is consistently lower than what is seen in gum arabic and larch AG. Although most side chains were short, long chains such as ß-1,6-galactohexaose chains were also observed in these vegetables. The results suggest a conserved ß-1,3;1,6-galactan structure in the AGP-subclass that distinguishes it from the gum arabic-subclass.

Strategies for reducing per-sample costs in target capture sequencing for phylogenomics and population genomics in plants.

The reduced cost of high-throughput sequencing and the development of gene sets with wide phylogenetic applicability has led to the rise of sequence capture methods as a plausible platform for both phylogenomics and population genomics in plants. An important consideration in large targeted sequencing projects is the per-sample cost, which can be inflated when using off-the-shelf kits or reagents not purchased in bulk. Here, we discuss methods to reduce per-sample costs in high-throughput targeted sequencing projects. We review the minimal equipment and consumable requirements for targeted sequencing while comparing several alternatives to reduce bulk costs in DNA extraction, library preparation, target enrichment, and sequencing. We consider how each of the workflow alterations may be affected by DNA quality (e.g., fresh vs. herbarium tissue), genome size, and the phylogenetic scale of the project. We provide a cost calculator for researchers considering targeted sequencing to use when designing projects, and identify challenges for future development of low-cost sequencing in non-model plant systems.

Molecular basis for antagonistic activity of anifrolumab, an anti-interferon-α receptor 1 antibody.

Anifrolumab (anifrolumab) is an antagonist human monoclonal antibody that targets interferon α receptor 1 (IFNAR1). Anifrolumab has been developed to treat autoimmune diseases and is currently in clinical trials. To decipher the molecular basis of its mechanism of action, we engaged in multiple epitope mapping approaches to determine how it interacts with IFNAR1 and antagonizes the receptor. We identified the epitope of anifrolumab using enzymatic fragmentation, phage-peptide library panning and mutagenesis approaches. Our studies revealed that anifrolumab recognizes the SD3 subdomain of IFNAR1 with the critical residue R(279). Further, we solved the crystal structure of anifrolumab Fab to a resolution of 2.3 Å. Guided by our epitope mapping studies, we then used in silico protein docking of the anifrolumab Fab crystal structure to IFNAR1 and characterized the corresponding mode of binding. We find that anifrolumab sterically inhibits the binding of IFN ligands to IFNAR1, thus blocking the formation of the ternary IFN/IFNAR1/IFNAR2 signaling complex. This report provides the molecular basis for the mechanism of action of anifrolumab and may provide insights toward designing antibody therapies against IFNAR1.