Long-Read NGS
TELL-Seq Barcode Linked-Reads: Long-Read Sequencing for All
• Accurate Long Reads up to 200kb on Illumina Platform
• Only commercially available and very cost-effective barcode linked-reads reagent platform
• Extremely low input amounts – as low as 0.5 ng HMW DNA
• Easy scalable 3 hours protocol, all in one PCR tube
• Applicable to any species, genome, metagenome, and project size
• Library Prep Kits for eukaryotic genomes and metagenomics
• Microbial Isolate Library Prep Kits for small genomes
• Protocols for amplicon– and hyb capture– based targeted long-range NGS
• Free software packages for data analysis
Long-range sequencing information is required for de novo genome assembly, haplotype phasing, structural variation detection, as well as for the deconvolution of microbial genomes from complex metagenomics samples. Current long-read sequencing technologies can provide valuable long-range information but at a high cost with low accuracy and high DNA input requirement.
Our partner Universal Sequencing Technology have developed the single-tube Transposase Enzyme Linked Long-read Sequencing (TELL-seqTM) technology ( Chen et al., Genome Research, June 2020 ), which enables a low-cost, high-accuracy and high-throughput short-read second-generation sequencer to generate up to 200 kb long-range sequencing information with as little as 0.5 ng input material. And all of this without the requirement for any additional costly devices.
The TELL-Seq Barcode Linked-Reads Solution:
In a PCR tube, millions of clonally barcoded beads are used to uniquely barcode long DNA molecules in an open bulk reaction without dilution and compartmentation. The barcode linked reads are used to successfully assemble genomes ranging from microbes to human. These linked-reads also generate mega-base-long phased blocks for haplotyping and provide a cost-effective tool for detecting structural variants in a genome, which are important to identify compound heterozygosity in recessive Mendelian diseases and discover genetic drivers and diagnostic biomarkers in cancers.
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(A) Diagram of TELL-seq library preparation procedure. In a 0.2-mL PCR tube, 0.1 ng to 5 ng genomic DNA was mixed with 3–10 million barcoded TELL beads and transpososomes for the clonal barcoding reaction. Genomic DNA fragments were captured on the barcoded TELL beads via connecting strand transfer complexes (STCs) to barcode oligos on the bead surface. A tagging between STCs by a second transpososome introduced a second priming site for library amplification. After breaking the STCs and washing the magnetic TELL beads, sequencing library molecules were amplified off beads with P5 and P7 adaptor sequences incorporated at the same time. The total library procedure took ∼3 h.
(B) TELL-seq library structure for Illumina sequencing systems. Index 1 comprises 18-base TELL-seq molecular barcode; Index 2 comprises 8-base barcode for sample indexing.
See also UST´s whitepaper “A Brief Introduction to Linked-Read Technology”
User Manuals for TELL-Seq Kits and Reagents
TELL-Seq Data Analysis Software Packages – Free Download
De novo Genome Assembly and ScaffoldingUsing the TELL-Seq™ WGS library prep kits and the free TELL-seq software packages you can sequence and assemble novel genomes of any size – small bacterial genomes, medium sized insect, plant, and fungi genomes, and large human, animal and plant genomes. If you really want to understand a new genome, including gene synteny, you must look at long-reads. TELL-Seq™ kits will turn an ordinary short-read 2nd generation DNA sequencer into a long-read 3rd generation DNA sequencer. TELL-Seq™ linked reads improve de novo genome assembly, and resolve repeat regions as well as structural variations and translocations. Compared with conventional long-read sequencing, TELL-Seq™ is simpler and more cost effective, and it requires hundreds or even thousands of times less DNA input than long-read sequencers which makes TELL-Seq™ the long-read solution for scarce and precious DNA samples. TELL-Seq™ can also be used as a scaffolding method for traditional long-read assembly to acquire more accurate de novo reference genomes than using long reads alone.
For genomes > 50Mb we offer the TELL-Seq Library Kits for Eukaryotic Genomes. The TELL-Seq Microbial Library Kits are specifically designed for microbial isolate samples with less than 50Mb genome size to prepare TELL-Seq™ libraries with the least cost.
Literature References:
High-quality genome assemblies for nine non-model North American Insect Species
Draft genome assemblies of the avian louse Brueelia nebulosa and its associates using long-read sequencing from an individual specimen
Genome Sequence of Lichtheimia ornata, an Emerging Opportunistic Mucorales Pathogen
Low-cost and highly efficient generation of near-complete bacterial pathogen genomes by TELL-Seq
Metagenomics: Decipher microbial genomes in complex mixed environmental and clinical samples
Samples containing disparate microbes are essential to gaining insight into our ecosystem as well as human disease. The TELL-Seq™ Library Prep kit for eukaryotic genomes and metagenomics will allow you to sequence samples at a metagenomic scale, providing long-read like results with the ease of short-read sequencing. To see what is truly in a mixed microbial sample use the power of long-read DNA sequencing with the simplicity of short-read sequencing.
Microbiome samples are complex and their contained genomes, which vary in abundance but overlap in composition, are particularly challenging to assemble. Current mainstream NGS platforms such as Illumina short reads lack the necessary read length for accurate de novo assembly of metagenomes. Commercially available long-read sequencing platforms are either too inaccurate for strain level variation or have too low throughput, and therefore are not able to satisfy the needs of many metagenomic studies.
The TELL-Seq™ Linked-Reads Metagenomics pipeline which includes the TELL-Seq Library Prep Kit and the free TELL-Meta analysis software is emerging as the ideal solution for metagenomics studies and enables de novo assemblies of the contained microbial genomes. TELL-Seq™ can identify species more accurately than short reads alone with far fewer false positives and more accurate abundance estimation and empowers discovery of new microbial species & strains and their genomes. TELL-Seq™ also generates high-quality MAGs (metagenome-assembled genomes) better than long-read platforms. TELL-Seq™ library prep is extremely cost-effective as well as easy to scale up and automate. Multiplex indices are available to allow pooling of hundreds of samples to sequence on high-throughput low-cost Illumina NGS sequencers.
Literature References:
Characterizing the microbial metagenome of calcareous stromatolite formations in the San Felipe Creek in Anza Borrego Desert
Understanding the impact of radical changes in diet and the gut microbiota on brain function and structure: rationale and design of the
EMBRACE study
Genome of a novel Sediminibacterium discovered in association with two species of freshwater cyanobacteria from streams in Southern California
Anoxygenic phototroph of the Chloroflexota uses a type I reaction centre
Please also visit our website featuring the iSWAB-Microbiome Collection System for collecting and storing microbiome samples at room temperature
Structural Variant Calling
Short-read DNA sequencers have trouble identifying large structural variants such as copy number variations (CNVs), translocations, duplications, deletions, insertions, and inversions. The TELL-Seq™ Library Prep Kits for Eukaryotic Genomes link short-reads turning them into long-reads. Using the free TELL-Sort software to analzye the linked, long-read data that you get using the TELL-Seq™ kit will allow you to take a step back and see what you were missing.
Literature References:
Human commensal Candida albicans strains demonstrate substantial within-host diversity and retained pathogenic potential demonstrated by TELL-Seq
Linked‑read sequencing for detecting short tandem repeat expansions
LRTK: a platform agnostic toolkit for linked-read analysis of both human genome and metagenome
• Whole Genome and Targeted Haplotype Phasing
Knowing which homologous chromosome has which sequence is essential to understanding a genome. Phasing provides haplotype information which is important for understanding complex traits and variant linkages. However, Whole Genome Phasing is difficult with 2nd generation, short-read, DNA sequencing technology. The TELL-Seq™ Library Prep Kit for Eukaryotic Genomes helps with this by linking short-reads to larger fragments and generate long-range information for both whole genome haplotyping or targeted phasing applications using the free TELL-Sort software.
Literature References:
SpLitteR: Diploid genome assembly using TELL-Seq linked-reads and assembly graphs
Illumina Application Note: “UST TELL-Seq using Illumina NGS platforms enables genome-scale haplotype”
OVERVIEW: TELL-Seq Publications and Data
Visit the FAQ site for detailed technical information about TELL-Seq
Please also visit our Hi-C Sequencing website.
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