Sequencing by binding rivals SMOR error-corrected sequencing by synthesis technology for accurate detection and quantification of minor (< 0.1%) subpopulation variants.

BACKGROUND: Detecting very minor (< 1%) subpopulations using next-generation sequencing is a critical need for multiple applications, including the detection of drug resistant pathogens and somatic variant detection in oncology. A recently available sequencing approach termed 'sequencing by binding (SBB)' claims to have higher base calling accuracy data "out of the box." This paper evaluates the utility of using SBB for the detection of ultra-rare drug resistant subpopulations in Mycobacterium tuberculosis (Mtb) using a targeted amplicon assay and compares the performance of SBB to single molecule overlapping reads (SMOR) error corrected sequencing by synthesis (SBS) data. RESULTS: SBS displayed an elevated error rate when compared to SMOR error-corrected SBS and SBB techniques. SMOR error-corrected SBS and SBB technologies performed similarly within the linear range studies and error rate studies. CONCLUSIONS: With lower sequencing error rates within SBB sequencing, this technique looks promising for both targeted and unbiased whole genome sequencing, leading to the identification of minor (< 1%) subpopulations without the need for error correction methods.

Engineering psychrophilic polymerase for nanopore long-read sequencing.

Unveiling the potential application of psychrophilic polymerases as candidates for polymerase-nanopore long-read sequencing presents a departure from conventional choices such as thermophilic Bacillus stearothermophilus (Bst) renowned for its limitation in temperature and mesophilic Bacillus subtilis phage (phi29) polymerases for limitations in strong exonuclease activity and weak salt tolerance. Exploiting the PB-Bst fusion DNA polymerases from Psychrobacillus (PB) and Bacillus stearothermophilus (Bst), our structural and biochemical analysis reveal a remarkable enhancement in salt tolerance and a concurrent reduction in exonuclease activity, achieved through targeted substitution of a pivotal functional domain. The sulfolobus 7-kDa protein (Sso7d) emerges as a standout fusion domain, imparting significant improvements in PB-Bst processivity. Notably, this study elucidates additional functional sites regulating exonuclease activity (Asp43 and Glu45) and processivity using artificial nucleotides (Glu266, Gln283, Leu334, Glu335, Ser426, and Asp430). By disclosing the intricate dynamics in exonuclease activity, strand displacement, and artificial nucleotide-based processivity at specific functional sites, our findings not only advance the fundamental understanding of psychrophilic polymerases but also provide novel insights into polymerase engineering.

Sequencing by Binding rivals error-corrected Sequencing by Synthesis technology for accurate detection and quantification of minor (<0.1%) subpopulation variants.

Detecting very minor (< 1%) subpopulations using next-generation sequencing is a critical need for multiple applications including detection of drug resistant pathogens and somatic variant detection in oncology. To enable these applications, wet lab enhancements and bioinformatic error correction methods have been developed for 'sequencing by synthesis' technology to reduce its inherent sequencing error rate. A recently available sequencing approach termed 'sequencing by binding' claims to have higher base calling accuracy data "out of the box." This paper evaluates the utility of using 'sequencing by binding' for the detection of ultra-rare subpopulations down to 0.001%.

Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform.

In Phi29-α-hemolysin (α-HL) nanopore sequencing systems, a strong electrochemical signal is dependent on a high concentration of salt. However, high salt concentrations adversely affect polymerase activity. Sequencing by synthesis (SBS) requires the use of phi29 polymerase without exonuclease activity to prevent the degradation of modified nucleotide tags; however, the lack of exonuclease activity also affects polymerase processivity. This study aimed to optimize phi29 polymerase for improved salt tolerance and processivity while maintaining its lack of exonuclease activity to meet the requirements of nanopore sequencing. Using salt tolerance compartmentalized self-replication (stCSR) and a microfluidic platform, we obtained 11 mutant sites with enhanced salt tolerance attributes. Sequencing and biochemical analyses revealed that the substitution of conserved amino acids such as G197D, Y369E, T372N, and I378R plays a critical role in maintaining the processivity of exonuclease-deficient phi29 polymerase under high salt conditions. Furthermore, Y369E and T372N have been identified as important determinants of DNA polymerase binding affinity. This study provides insights into optimizing polymerase processability under high-salt conditions for real-time polymerase nanopore sequencing, paving the way for improved performance and applications in nanopore sequencing technologies.

Phenanthrene Degradation by Photosynthetic Bacterial Consortium Dominated by Fischerella sp.

Microbial degradation of aromatic hydrocarbons is an emerging technology, and it is well recognized for its economic methods, efficiency, and safety; however, its exploration is still scarce and greater emphasis on cyanobacteria-bacterial mutualistic interactions is needed. We evaluated and characterized the phenanthrene biodegradation capacity of consortium dominated by Fischerella sp. under holoxenic conditions with aerobic heterotrophic bacteria and their molecular identification through 16S rRNA Illumina sequencing. Results indicated that our microbial consortium can degrade up to 92% of phenanthrene in five days. Bioinformatic analyses revealed that consortium was dominated by Fischerella sp., however different members of Nostocaceae and Weeksellaceae, as well as several other bacteria, such as Chryseobacterium, and Porphyrobacter, were found to be putatively involved in the biological degradation of phenanthrene. This work contributes to a better understanding of biodegradation of phenanthrene by cyanobacteria and identify the microbial diversity related.

Assessment of the ForenSeq mtDNA control region kit and comparison of orthogonal technologies.

The ForenSeq® mtDNA Control Region Kit, MiSeq FGx®, and Universal Analysis Software (UAS) were assessed to better define the performance and limitations of the system with forensically relevant samples to provide data for its transition into practice. A total of six MiSeq FGx sequencing runs of ForenSeq mtDNA Control Region kit, three runs of additional orthogonal sequencing chemistries, and Sanger sequencing results for 14 samples were used to test for concordance. Sensitivity, reproducibility, mixture detection studies, as well as studies to measure the performance of amplification and sequencing controls were performed. The use and reliability of the UAS for data analysis was also examined. With a variety of sample types and controls representing many mitochondrial haplotypes, the recently developed mtDNA Control Region Kit, with the MiSeq FGx and UAS, was found to be fit for purpose as reliable, reproducible, and robust. Sensitivity down to 1 pg of input genomic DNA was demonstrated, which allows the system to offer low limits of detection for better interrogation of potential heteroplasmy in samples. Concerns for implementing next generation sequencing (NGS) for mtDNA in laboratories were addressed in this research, including initial template quantification and confirmation of haplotypes generated by UAS software regarding length-based polymorphisms. To improve performance with forensic samples, laboratories could implement mitochondrial-specific qPCR assays for quantification and perform the optional manual normalization protocol. Additional optimization on sample multiplexing can provide methods that either increase sensitivity or cost efficiency of the assay.

Genomic Resources for Salminus brasiliensis.

The Neotropical region bears the most diverse freshwater fish fauna on the planet and is the stage for dramatic conservation struggles. Initiatives aiming for conservation of a single emblematic fish, a flagship species, to which different onlookers relate on a cultural/personal level, holds promise towards engagement and conservation actions benefiting whole biological communities and ecosystems. Here, we present the first comprehensive genomic resources for Salminus brasiliensis, a potential flagship Neotropical species. This fish faces pressing conservation issues, as well as taxonomic uncertainty, being a main species relevant to angling and commercial fisheries. We make available 178 million Illumina paired-end reads, 90 bases long, comprising 16 Gb (≈15X coverage) of filtered data, obtained from a primary genomic library of 500-bp fragments. We present the first de novo genomic assembly for S. brasiliensis, with ∼1 Gb (N 50 = 10,889), as well as the coding genome annotation of 12,962 putative genes from assembled genomic fragments over 10 kb, most of which could be identified from the Ostariophysi GenBank database. We also provide a genome-wide panel for more than 80,000 predicted microsatellite loci for low-cost, fast and abundant DNA marker development for this species. A total of 47, among 52 candidates, empirically assayed microsatellites were confirmed as polymorphic in this fish. All genomic data produced for S. brasiliensis is hereby made publicly accessible. With the disclosure of these results, we intend to foster general biology studies and to provide tools to be applied immediately in conservation and aquaculture in this candidate flagship Neotropical species.

Pyrosequencing Primer Design for Forensic Biology Applications.

The polymerase chain reaction (PCR) is used to copy DNA in vitro for a variety of applications including amplifying a target DNA, mutating a base, adding tags, and sequencing by synthesis applications. Next-generation sequencing (NGS) is a DNA sequencing technology that has been applied to screening cancer and tissue variants, deep sequencing, and gene expression analysis, and more recently, it has been applied to DNA typing for human identification, estimating age, and detecting and differentiating body fluids. Body fluids are normally identified using color tests, microscopy, and immunochromatographic assays. Pyrosequencing is an NGS approach that has been applied to body fluid analysis. The pyrosequencing assays can detect one or several mixed body fluids by analysis of their tissue-specific differentially methylated regions (tDMRs). Here, the process of designing pyrosequencing primers for forensic biology applications is described.

A virtual sequencer reveals the dephasing patterns in error-correction code DNA sequencing.

An error-correction code (ECC) sequencing approach has recently been reported to effectively reduce sequencing errors by interrogating a DNA fragment with three orthogonal degenerate sequencing-by-synthesis (SBS) reactions. However, similar to other non-single-molecule SBS methods, the reaction will gradually lose its synchronization within a molecular colony in ECC sequencing. This phenomenon, called dephasing, causes sequencing error, and in ECC sequencing, induces distinctive dephasing patterns. To understand the characteristic dephasing patterns of the dual-base flowgram in ECC sequencing and to generate a correction algorithm, we built a virtual sequencer in silico. Starting from first principles and based on sequencing chemical reactions, we simulated ECC sequencing results, identified the key factors of dephasing in ECC sequencing chemistry and designed an effective dephasing algorithm. The results show that our dephasing algorithm is applicable to sequencing signals with at least 500 cycles, or 1000-bp average read length, with acceptably low error rate for further parity checks and ECC deduction. Our virtual sequencer with our dephasing algorithm can further be extended to a dichromatic form of ECC sequencing, allowing for a potentially much more accurate sequencing approach.

Human Mitochondrial Control Region and mtGenome: Design and Forensic Validation of NGS Multiplexes, Sequencing and Analytical Software.

Forensic mitochondrial DNA (mtDNA) analysis conducted using next-generation sequencing (NGS), also known as massively parallel sequencing (MPS), as compared to Sanger-type sequencing brings modern advantages, such as deep coverage per base (herein referred to as read depth per base pair (bp)), simultaneous sequencing of multiple samples (libraries) and increased operational efficiencies. This report describes the design and developmental validation, according to forensic quality assurance standards, of end-to-end workflows for two multiplexes, comprised of ForenSeq mtDNA control region and mtDNA whole-genome kits the MiSeq FGxTM instrument and ForenSeq universal analysis software (UAS) 2.0/2.1. Polymerase chain reaction (PCR) enrichment and a tiled amplicon approach target small, overlapping amplicons (60-150 bp and 60-209 bp for the control region and mtGenome, respectively). The system provides convenient access to data files that can be used outside of the UAS if desired. Studies assessed a range of environmental and situational variables, including but not limited to buccal samples, rootless hairs, dental and skeletal remains, concordance of control region typing between the two multiplexes and as compared to orthogonal data, assorted sensitivity studies, two-person DNA mixtures and PCR-based performance testing. Limitations of the system and implementation considerations are discussed. Data indicated that the two mtDNA multiplexes, MiSeq FGx and ForenSeq software, meet or exceed forensic DNA quality assurance (QA) guidelines with robust, reproducible performance on samples of various quantities and qualities.

Mikan Genome Database (MiGD): integrated database of genome annotation, genomic diversity, and CAPS marker information for mandarin molecular breeding.

Citrus species are some of the most valuable and widely consumed fruits globally. The genome sequences of representative citrus (e.g., Citrus clementina, C. sinensis, C. grandis) species have been released but the research base for mandarin molecular breeding is still poor. We assembled the genomes of Citrus unshiu and Poncirus trifoliata, two important species for citrus industry in Japan, using hybrid de novo assembly of Illumina and PacBio sequence data, and developed the Mikan Genome Database (MiGD). The assembled genome sizes of C. unshiu and P. trifoliata are 346 and 292 Mb, respectively, similar to those of citrus species in public databases; they are predicted to possess 41,489 and 34,333 protein-coding genes in their draft genome sequences, with 9,642 and 8,377 specific genes when compared to C. clementina, respectively. MiGD is an integrated database of genome annotation, genetic diversity, and Cleaved Amplified Polymorphic Sequence (CAPS) marker information, with these contents being mutually linked by genes. MiGD facilitates access to genome sequences of interest from previously reported linkage maps through CAPS markers and obtains polymorphism information through the multiple genome browser TASUKE. The genomic resources in MiGD (https://mikan.dna.affrc.go.jp) could provide valuable information for mandarin molecular breeding in Japan.

Massive parallel sequencing in forensics: advantages, issues, technicalities, and prospects.

In the last decade, next-generation sequencing (NGS) technology, alternatively massive parallel sequencing (MPS), was applied to all fields of biological research. Its introduction to the field of forensics was slower, mainly due to lack of accredited sequencers, kits, and relatively higher sequencing error rates as compared with standardized Sanger sequencing. Currently, a majority of the problematic issues have been solved, which is proven by the body of reports in the literature. Here, we discuss the utility of NGS sequencing in forensics, emphasizing the advantages, issues, the technical aspects of the experiments, commercial solutions, and the potentially interesting applications of MPS.

Long walk to genomics: History and current approaches to genome sequencing and assembly.

Genomes represent the starting point of genetic studies. Since the discovery of DNA structure, scientists have devoted great efforts to determine their sequence in an exact way. In this review we provide a comprehensive historical background of the improvements in DNA sequencing technologies that have accompanied the major milestones in genome sequencing and assembly, ranging from early sequencing methods to Next-Generation Sequencing platforms. We then focus on the advantages and challenges of the current technologies and approaches, collectively known as Third Generation Sequencing. As these technical advancements have been accompanied by progress in analytical methods, we also review the bioinformatic tools currently employed in de novo genome assembly, as well as some applications of Third Generation Sequencing technologies and high-quality reference genomes.

Enzymatic Synthesis of Designer DNA Using Cyclic Reversible Termination and a Universal Template.

Phosphoramidite chemistry remains the industry standard for DNA synthesis despite significant limitations on the length and yield of the oligonucleotide, time restrictions, and hazardous waste production. Herein, we demonstrate the synthesis of single-stranded oligos on a solid surface by DNA polymerases and reverse transcriptases. We report the extension of surface-bound oligonucleotides enabled by transient hybridization of as few as two bases to a neighboring strand. When multiple hybridization structures are possible, each templating a different base, a DNA polymerase or reverse transcriptase can extend the oligonucleotide with any of the complementary bases. Therefore, the sequence of the newly synthesized fragment can be controlled by adding only the desired base as a substrate to the reaction solution. We used this enzymatic approach to synthesize a 20 base oligonucleotide by incorporating reversible terminator dNTPs through a two-step cyclic reversible termination process with a corrected stepwise efficiency over 98%. In our approach, a nascent DNA strand that serves as both primer and template is extended through polymerase-controlled sequential addition of 3'-reversibly blocked nucleotides followed by subsequent cleavage of the 3'-capping group. This process enables oligonucleotide synthesis in an environment not permitted by traditional phosphoramidite methods, eliminates the need for hazardous chemicals, has the potential to provide faster and higher yield results, and synthesizes DNA on a solid support with a free 3' end.

Transcriptomic data on the role of PEST-domain-enriched tyrosine phosphatase in the regulation of antigen-mediated activation and antiallergic action of glucocorticoids in mast cells.

Protein tyrosine phosphatases and glucocorticoids are known to regulate allergic and antiallergic action in activated mast cells. Here we provide RNA sequencing and quantitative real-time PCR data from bone marrow derived mast cells, for wild-type and PEST-domain-enriched tyrosine phosphatase (PEP) null mice, activated by immunoglobulin E sensitization and dinitrophenol treatment, and additionally treated with the glucocorticoid dexamethasone. The transcriptomics experiment was performed in duplicate with a total of 16 samples (GSE108972).

Genome Sequencing.

Strategies for sequencing fungal genomes on next-generation sequencing (NGS) platforms depend on the characteristics of the genome of the targeted species, quantity and quality of the genomic DNA, and cost considerations. Massively parallel sequencing with sequencing by synthesis (SBS) approach by Illumina produces terabases of short read sequences (i.e., ~300 bp) in a time and cost-effective manner, though the read length can limit the assembly particularly in repetitive regions. The single molecule, real-time (SMRT) sequencing approach by Pacific Biosciences (PacBio) produces longer reads (i.e., ~12,500 bp) which can facilitate de novo assembly of genomes that contain long repetitive sequences, though due to the lower-throughput of this platform achieving the coverage needed for assembly is more expensive than by SBS. Additionally, the Illumina SBS platforms can handle low quantity/quality of genomic DNA materials, while the SMRT system requires undamaged long DNA fragments as input to ensure that high-quality data is produced. Both platforms are discussed in this chapter including key decision-making points.

MiSeq: A Next Generation Sequencing Platform for Genomic Analysis.

MiSeq, Illumina's integrated next generation sequencing instrument, uses reversible-terminator sequencing-by-synthesis technology to provide end-to-end sequencing solutions. The MiSeq instrument is one of the smallest benchtop sequencers that can perform onboard cluster generation, amplification, genomic DNA sequencing, and data analysis, including base calling, alignment and variant calling, in a single run. It performs both single- and paired-end runs with adjustable read lengths from 1 × 36 base pairs to 2 × 300 base pairs. A single run can produce output data of up to 15 Gb in as little as 4 h of runtime and can output up to 25 M single reads and 50 M paired-end reads. Thus, MiSeq provides an ideal platform for rapid turnaround time. MiSeq is also a cost-effective tool for various analyses focused on targeted gene sequencing (amplicon sequencing and target enrichment), metagenomics, and gene expression studies. For these reasons, MiSeq has become one of the most widely used next generation sequencing platforms. Here, we provide a protocol to prepare libraries for sequencing using the MiSeq instrument and basic guidelines for analysis of output data from the MiSeq sequencing run.

Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array.

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5'-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.

Illumina error profiles: resolving fine-scale variation in metagenomic sequencing data.

BACKGROUND: Illumina's sequencing platforms are currently the most utilised sequencing systems worldwide. The technology has rapidly evolved over recent years and provides high throughput at low costs with increasing read-lengths and true paired-end reads. However, data from any sequencing technology contains noise and our understanding of the peculiarities and sequencing errors encountered in Illumina data has lagged behind this rapid development. RESULTS: We conducted a systematic investigation of errors and biases in Illumina data based on the largest collection of in vitro metagenomic data sets to date. We evaluated the Genome Analyzer II, HiSeq and MiSeq and tested state-of-the-art low input library preparation methods. Analysing in vitro metagenomic sequencing data allowed us to determine biases directly associated with the actual sequencing process. The position- and nucleotide-specific analysis revealed a substantial bias related to motifs (3mers preceding errors) ending in "GG". On average the top three motifs were linked to 16 % of all substitution errors. Furthermore, a preferential incorporation of ddGTPs was recorded. We hypothesise that all of these biases are related to the engineered polymerase and ddNTPs which are intrinsic to any sequencing-by-synthesis method. We show that quality-score-based error removal strategies can on average remove 69 % of the substitution errors - however, the motif-bias remains. CONCLUSION: Single-nucleotide polymorphism changes in bacterial genomes can cause significant changes in phenotype, including antibiotic resistance and virulence, detecting them within metagenomes is therefore vital. Current error removal techniques are not designed to target the peculiarities encountered in Illumina sequencing data and other sequencing-by-synthesis methods, causing biases to persist and potentially affect any conclusions drawn from the data. In order to develop effective diagnostic and therapeutic approaches we need to be able to identify systematic sequencing errors and distinguish these errors from true genetic variation.

Development of nuclear and chloroplast microsatellite markers for the endangered conifer Callitris sulcata (Cupressaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for Callitris sulcata (Cupressaceae), an endangered conifer species in New Caledonia. METHODS AND RESULTS: Using sequencing by synthesis (SBS) of an RNA-Seq library, 15 polymorphic nuclear and chloroplast microsatellite markers were developed. When evaluated with 48 individuals, these markers showed genetic variations ranging from two to 15 alleles and expected heterozygosity ranging from 0 to 0.881. CONCLUSIONS: These markers will be useful for examining the genetic diversity and structure of remaining wild populations and improving the genetic status of ex situ populations.