Next-generation sequencing (NGS) is a massively parallel sequencing technology that performs millions to billions of individual sequencing reactions simultaneously in a process. NGS greatly improves sequencing output compared to Sanger sequencing while being more time-saving and cost-effective. Most of the currently available next-generation technologies are based on the sequencing by synthesis approach. The second- and third- generation sequencing methods are defined as short-read and long-read technologies, respectively. Despite the vast differences in NGS methods, the workflow mainly follows three steps, including sample preparation, nucleic acid sequencing, and data analysis.
Library preparation is the first step of NGS, which is the preparation of nucleic acid template (DNA or RNA) for sequencing. For short-read sequencing applications, isolated nucleic acids need to be fragmented, end repaired, and covalently attached to adapters using ligation or tagmentation methods. Then templates are amplified so that there are enough copies of each template for the sequencer to detect them. Long-read sequencing platforms differ from short-read sequencing platforms in library preparation in that no amplification step is required and, in some long-read platforms, original DNA or RNA molecules are directly sequenced. The key goal of library preparation is to maximize complexity while minimizing bias introduced by PCR or other amplifications, as the quality of the resulting library can strongly influence NGS results.
Amerigo Scientific offers NGS library preparation tools based on Transposase Enzyme-Linked Long-read Sequencing (TELL-Seq™) technology to simplify workflows and facilitate the preparation of high quality DNA libraries. With TELL-Seq™ library prep kits, sequencing-ready libraries can be prepared quickly and cost-effectively in about 3 hours with a simple workflow and only basic lab equipment.
TELL-Seq™ Bundles, Boxes and Kits
The advanced TELL-Seq™ library preparation kits enable short-read (second-generation) sequencing platforms such as Illumina and MGI to produce results averaging 20 Kb to 200 Kb with only 3 hours of library preparation and minimal DNA input material.
The TELL-Seq™ recommended bundles are available in both standard and high-throughput bundles.
The TELL-Seq™ standard bundle has enough reagent to prepare 4 sample libraries for genomes 1 Gb in size or larger or 12 sample libraries for small genomes up to 200 Mb in size for both diploid and haploid genomes.
The TELL-Seq™ HT24 high-throughput bundle has enough reagent to prepare 24 sample libraries of 1 Gb or larger in size or 72 sample libraries up to 200 Mb in size for both diploid and haploid genomes.
To be used with the TELL-Seq™ WGS Library Prep Kit for targeted applications
Features
Long-read NGS library preparation method
Ease of use: hands-on time ( < 1 hour)
Single tube process: sequencing ready library in 3 hours
Highly scalable for small and large genomes
Easily automated
Low DNA input: 0.5 ng to 5 ng depending on genome size
Applications
De novo sequencing (small and large genomes)
Microbiology and metagenomics
Whole human genome phasing
Whole exome and target phasing
Structure variation detection
TELL-Seq™ Technology
TELL-Seq™ technology is a transposase-based linked-read library technology that generates barcoded-linked reads for genome-scale sequencing applications on short-read sequencers. The whole procedure can be carried out in a PCR tube without the need for expensive instrumentation. Inside the PCR tube, millions of clonally barcoded magnetic beads are added to the sample and interact with the DNA molecules. Each bead contains a unique 18 bp barcode sequence conjugated onto the surface. This allows them to uniquely barcode long DNA molecules and to assemble genomes ranging from the size of a microbe to a human. These linked reads also generate megabase-long phased blocks and provide a cost-effective tool for detecting structural variants, which are essential to identifying compound heterozygosity in recessive Mendelian diseases and interpretation of genome-wide association studies (GWAS), discovering genetic drivers, and diagnosing genetic biomarkers.
TELL-Seq™ Library Preparation and Sequencing Workflow
TELL-Seq™ Library Preparation Principle
TELL-Seq™ Library Structure and Sequencing Scheme
TELL-Seq™ Applications
Whole Genome Sequencing
Whole-genome sequencing (WGS) enables mapping and de novo assembly of novel organisms, completing genomes of known organisms, and comparing genomes between samples. Short-read sequencing platforms such as Illumina® are highly accurate and cost-effective for many applications, but are limited by sequences ≤500 to 600 bp per read. For de novo sequencing, where the reference genome is not available for alignment and reads are assembled into contigs, TELL-Seq™ enables Illumina® NGS platforms to generate highly accurate data while reducing cost, turnaround time, and DNA input requirements. This makes TELL-Seq™ an ideal solution for applications such as highly polished de novo assembly of small genomes.
Metagenomics
Understanding microbial diversity at the strain level is critical to the study of human health and disease. Metagenomics is the study of genetic material recovered directly from environmental samples. It is often used to study a specific community of microorganisms and enables the characterization of microbial diversity in clinical, food, and environmental samples. When recovering and annotating genomes from metagenomic sequencing data, assembling a genome without the use of references becomes complicated. The presence of multiple organisms in the microbial mixture that vary widely in abundance, relatedness, diversity, and repetitive content can greatly complicate the genome assembly process. Tell-TaxContigs is a computational pipeline that enables taxonomic identification of bacterial and archaea species and estimates their relative abundance. Tell-TaxContigs uses pre-assembled metagenomic data generated by the TELL-Seq™ Library Prep kit and Tell-Link (a software for fragment assembly). With the long-range resolution generated by TELL-Seq™ linked reads, de novo assembly with Tell-Link software enables highly contiguous genome assemblies of microbial isolates.
Human Whole Genome Phasing
Each human genome contains maternal and paternal copies of each chromosome, and both copies are homologous in autosomal chromosomes except for positions containing heterozygous variants. Illumina® driven NGS has paved the way for the massive growth in annotating heterozygous variants. However, many clinical and research applications require resolving multivariant sets by chromosomal copy (haplotypes), a process known as phasing.
Illumina® NGS relies on short sequencing reads, which does not allow phasing beyond a few hundred bases using standard DNA library preparation kits. The use of the TELL-Seq™ Library Prep kit can increase long-read phasing capabilities of short-read sequencing instruments such as Illumina. TELL-Seq™ captures long-range information in short reads during NGS library preparation which enables the rapid and efficient phasing of entire genomes with high-molecular-weight (HMW) DNA of 50 to 200 kb.
Structural Variations Detection
Long-range sequencing information is required to identify structural variations (SVs). As a simple, scalable, linked-read library preparation technology, TELL-Seq™ enables de novo assembly and can be used as an alternative method to resolve haplotypes when a reference genome is lacking or when a sizeable structural variant is present.
Phased Whole Exome and Targeted Sequencing
Each human genome has 4-5 million sites on all chromosomes that differ from the reference human genome. However, most sequence differences (called alternate alleles) are found only in either the maternal copy or paternal copy (known as heterozygous) of each chromosome. Determining whether alternate alleles from different loci are on the same or different chromosomal molecules, a process known as haplotype staging, is critical for many research and clinical applications.
Widely used short-read sequencing technologies produce highly accurate but relatively short reads. Short reads rarely span more than one heterozygous site because of the relatively low frequency of these positions. Long-read sequencing platforms enable phasing on heterozygous sites located kilobases apart, but with relatively lower sensitivity and fidelity or higher cost. The TELL-Seq™ Library Prep kit enables haplotype phasing with the accuracy of Illumina sequencing. TELL-Seq™ can phase relatively short DNA fragments, such as PCR products, in the 2 kb to 15 kb range.
TELL-Seq™ Application Examples
Results I: IGV Snapshot of TELL-Seq Linked Reads
Results II: TELL-Seq Results for Three Bacteria with Different
Results III: Novo Assembly Results Compared with Reference
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