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IDT

NGS megoldások az IDT-től!

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Az Integrated DNA Technologies természetesen a modern tudomány egyik legdinamikusabban növekvő ágában, az újgenerációs szekvenálásban is számtalan termékkel tudja segíteni az Ön vizsgálatait!

Az alábbiakban néhány példát válogattunk össze az NGS portfólióból (Adapterek, Library Prep Kit, Exome Research Panel).

A teljes portfólióért látogasson el az IDT Next Generation Sequencing weboldalára:
https://eu.idtdna.com/pages/products/next-generation-sequencing

ÁRAJÁNLATOT, INFORMÁCIÓT KÉREK!

https://eu.idtdna.com/pages/products/next-generation-sequencing

Adapters for next generation sequencing

High quality adapters that meet the ever-expanding needs of researchers using NGS

Adapters are a key component of the next generation sequencing (NGS) workflow. Whether your project requires a basic adapter or a sophisticated design for higher accuracy, IDT has the products and expertise to deliver the right solution.

Regardless of the NGS instrument or application, IDT has been serving the needs of NGS scientists for over 10 years. We are recognized as the leader in adapter synthesis based on our experience in custom oligo manufacturing and our commitment to quality. Additionally, our NGS customers and partners benefit from:

  • A comprehensive adapter offering
  • Innovative designs
  • Trusted customer support
  • Complete customizability

Due to our leadership in NGS adapter synthesis, Illumina chose IDT as its partner to develop the next generation of index adapters to improve sample multiplexing. These adapters contain Illumina’s unique dual indexes (UDIs) that mitigate sample misassignment due to index hopping. IDT manufactures UDI adapters for Illumina and is also licensed to sell your own custom designed adapters containing these UDI sequences. See the press release here.

Options for sequencing adapters with molecular barcodes.

Figure 1. Examples of adapter designs. A variety of NGS adapter designs are available. When selecting or designing adapters, consideration must be given to the intended application, multiplexing needs, accuracy requirements, and analysis methods.

Considerations when selecting an adapter design

IDT provides xGen Dual Index UMI Adapters, xGen Stubby Adapter and UDI Primer Pairs, and a Custom Adapter Configurator tool that guides you through design of Custom NGS Adapters. Below we describe some of the design considerations you will want to take into account.

Sequences for specific NGS platforms: During library preparation, adapters are attached (by ligation, PCR, or tagmentation) to the fragments of each sample library. Adapters include platform-specific sequences for fragment recognition by the sequencer: for example, the P5 and P7 sequences (Figure 1) enable library fragments to bind to the flow cells of Illumina platforms. Each NGS instrument provider uses a specific set of sequences for this purpose. IDT manufactures adapters for all major NGS platforms.

Sample indexing: Sample indexes (or indices) enable multiple samples to be sequenced together (i.e., multiplexed) on the same instrument flow cell or chip. Each sample index, typically 6–10 bases, is specific to a given sample library and is used for de-multiplexing during data analysis to assign individual sequence reads to the correct sample. Adapters may contain single or dual sample indexes depending on the number of libraries combined and the level of accuracy desired. Illumina recommends using unique dual indexes (UDIs) as a method to mitigate errors introduced by index-hopping. UDIs are particularly important when using instruments with patterned flow cells, such as the NovaSeq system.

IDT provides several indexing options with its Custom NGS Adapters. These include both IDT and Illumina 8 and 10 base index series, as well as the ability to upload your own index files.

Molecular barcoding: Unique molecular identifiers (UMIs) provide the highest levels of error correction and accuracy. UMIs are short sequences, often with degenerate bases, that incorporate a unique barcode onto each molecule within a given sample library. UMIs have been shown to reduce the rate of false-positive variant calls and increase sensitivity of variant detection. By incorporating individual barcodes on each original DNA fragment, variant alleles present in the original sample (true variants) can be distinguished from errors introduced during library preparation, target enrichment, or sequencing. Any identified errors can be removed by bioinformatics methods before final data analysis. Adapters that contain UMIs, such as the xGen Dual Index UMI Adapters, are available with a UDI design for detection of low-frequency variants. IDT Custom NGS Adapters can also be configured with UMIs.

Adapter manufacturing: Stringent manufacturing methods are critical for producing high quality NGS adapters. Substandard manufacturing practices can lead to low purity adapters or adapter cross contamination, either of which will negatively impact sequencing results. The IDT proprietary TruGrade process uses state-of-the-art synthesis and purification methods, designed specifically for NGS adapters. IDT also offers GMP grade adapter manufacturing for clinical applications.

Choose IDT adapter products based on the flexibility your experimental workflow requires. Click on the xGen Dual Index UMI Adapters—Tech Access, the xGen Stubby Adapter and UDI Primer Pairs, or the Custom NGS Adapters headings in Table 1 to review product offerings. The Custom NGS Adapters provide a step-by-step tool to help you configure adapters best suited for your research.

SpecificationsxGen Dual Index UMI Adapters—Tech AccessxGen Stubby Adapter and UDI Primer PairsCustom NGS adapters
Configuration UDI-UMI UDI single index, combinatorial dual indexing, UDI, or UDI-UMI
Sample index length 8 base, non-redundant sample indexes 8 base, non-redundant sample indexes Configurable
Concentration 15 µM  15 µM (Stubby Adapter), 20 µM total (10 µM each Primer) 15 µM (Adapter), 20 µM total (10 µM each Primer)
Shipping container Tubes, plates  Tubes (Stubby Adapter), 96-well plates (UDI Primer Pairs) Tubes, plates, or single-use plates
Reactions Varies 1 reaction per primer pair Varies
Adapter modifications Available Methylated for bisulfite sequencing* Available
Illumina platform compatibility 2- and 4-color platforms 2- and 4-color platforms Designs for any application

* For bisulfite sequencing, see our FAQ for additional information.

 


Lotus DNA Library Prep Kit

One kit. Flexible workflow. Endless applications.

The Lotus DNA Library Prep Kit enables streamlined preparation of high-quality next generation sequencing (NGS) libraries from double-stranded DNA (dsDNA). The kit uses enzymatic fragmentation to generate libraries suitable for PCR-free, PCR-amplified, and targeted sequencing applications on Illumina platforms.

  • Get the uniform sample coverage you need without relying on expensive equipment
  • Regain valuable time with a fast, simple workflow
  • Create application-specific NGS libraries by adding IDT adapters and xGen products for target capture

The Lotus kit can be customized for your applications when combined with one of the many IDT adapter options (see Ordering section). Additionally, use of xGen hybridization capture products provides a complete NGS solution that takes you from sample preparation to sequencing.

Applications

The Lotus DNA Library Prep Kit produces high-quality next generation sequencing (NGS) libraries that are suitable for many applications including:

  • Whole genome sequencing (WGS)
  • PCR-free sequencing
  • Detection of germline inherited single nucleotide variations (SNVs) and insertions/deletions (indels)
  • Hybridization capture of target sequences (e.g., the exome or transcripts of interest)
  • Low frequency somatic variation detection of SNVs and indels
  • RNA-seq starting with full-length, double-stranded cDNA input
  • Metagenomic sequencing

Method

This kit involves minimal enzymatic incubations and bead-based purification steps, thereby reducing sample handling and overall library preparation time to under 2 hours. The major steps are depicted in Figure 1 and are summarized as follows:

  • Enzymatic preparation. Fragmentation, end-repair, and dA-tailing of dsDNA are all performed in a single reaction.
  • Ligation of either full-length or stubby P5 and P7 adapters. When using full-length adapters, the final PCR step is optional and can be used to increase library yield. However, stubby adapters (sometimes called truncated adapters) require amplification with primers to incorporate sample indexing sequences and to add the P5 and P7 sequences.
  • Optional PCR amplification. Choose to amplify your libraries based on adapter and DNA input used. A bead-based cleanup removes oligonucleotides and small fragments after PCR.
Lotus library prep: time & main steps

Figure 1. Overview of Lotus DNA library preparation. Our easy enzymatic method takes you from sample to sequencing while eliminating the need for acoustic shearing methods that require instrumentation and extra time.

Kit overview

The Lotus kit comes with all the buffers, reagents, and enzymes needed for fragmentation, adapter ligation, and an optional PCR (to add index sequences, depending on adapter type, or to increase the amount of DNA for sequencing). You supply the DNA and add adapters that fit your goals. Table 1 provides additional specifications for using this kit.

Table 1. Specifications of the Lotus DNA Library Prep Kit.

FeatureDetails
Kit sizes 16 reactions
96 reactions

Sample types
(not provided)

Double-stranded DNA from fresh, frozen tissue
Genomic DNA
Full-length, double-stranded cDNA
DNA input range 1–250 ng
Suggested DNA insert size Whole-genome applications: 350 bp
Hybridization capture: 200 bp
TA-ligation adapters
(see Table 2)
Full-length: PCR amplification is optional
Stubby (truncated): PCR is required to add index sequences

Adapters

Complete your library prep with our selection of ready-to-use xGen Dual Index UMI Adapters or xGen Stubby Adapter and UDI Primer Pairs (Table 2). We also offer many high-quality, custom adapters and index primers that are application-specific and ideal for use with the Lotus kit.

Table 2. How to choose IDT adapters for common applications when using the Lotus DNA Library Prep Kit.

ApplicationBest choiceRationale
Whole genome sequencing (WGS), metagenomics, PCR-free sequencing (≥100 ng input) xGen Dual Index UMI Adapters–Tech Access Full-length adapters – required for PCR-free applications
WGS, metagenomics with PCR (1–250 ng input), exome sequencing, and targeted germline sequencing (SNVs, indels) xGen Stubby Adapter and UDI Primer Pairs Simple workflow – prepare your ligation master mix with a universal adapter and introduce unique dual index sample barcodes via PCR              
Low-level mutation detection, down to ~1% frequency xGen Dual Index UMI Adapters–Tech Access More sensitive – use UMI consensus analysis to remove sequencing errors
RNA-seq starting with full-length, double-stranded cDNA input xGen Dual Index UMI Adapters–Tech Access More quantitative – UMI is used to remove PCR duplicates

Complete workflow for hybridization capture experiments

Lotus DNA Library Prep Kit paired with adapters from IDT create libraries that are compatible with xGen hybridization capture probes and reagents for when you only need to perform targeted sequencing (Figure 2). Here is a list of the key components you will need:

  • xGen Hybridization and Wash Kit
  • xGen Universal Blockers—TS Mix or 10 bp TS Mix
  • xGen Lockdown Panels and Probe Pools
  • xGen Gene Capture Pools
  • xGen Library Amplification Primers
Lotus-xGen hybridization capture workflow
Figure 2. Overview of hybridization capture sequencing workflow.

Applikációk

xGen Exome Research Panel v2

Go ahead and commit. We made a lot.

We designed this updated panel using our new target-aware algorithm, resulting in the most complete coverage of any exome panel on the market. We assessed each probe with proprietary off-target analysis and manufactured them under strict ISO 13485 standards. We performed electrospray ionization mass spectrometry (ESI-MS) and dual quantification on each of the 415,115 probes before pooling. Finally, individual probe synthesis means we can deliver on a much larger scale than array methods, and produce a single large lot that can be aliquoted and stored securely, for years of reproducibility.

The xGen Exome Research Panel v2 gives you the confidence that only individual probe synthesis and individual QC can provide, and it comes at a price that makes your choice easy.

  • The new standard for exome coverage
  • Consistent performance through time
  • Infinite possibilities for customization
  • Time and cost savings
 

Ordering

Product details

Exome sequencing sequences only the protein-coding regions of the genome. Since exons represent only 1% of the human genome, an effective method of separating these regions from noncoding DNA is critical to focus on potentially important mutations implicated in disease. The captured material must also be suitable for sequencing to a satisfactory depth of coverage for reliable detection of variant alleles and sensitive applications like copy number variation (CNV) detection. To provide increased depth of coverage and enable high multiplexing of samples, the xGen Exome Research Panel v2 targets only the coding sequences (CDS) of human coding genes in the RefSeq 109 database.

xGen Exome Research Panel v2 consists of 5′ biotin–modified oligonucleotide probes that are individually synthesized and individually analyzed by ESI-MS and optical density (OD) measurement. The probes are normalized before pooling to ensure that each probe is represented in the panel at the correct concentration. Probes that fail quality control are resynthesized. This rigorous manufacturing process gives xGen Exome Research Panel v2 a unique advantage over array-derived pools in which missing or truncated probes cannot be identified before sequencing. IDT proprietary synthesis methods enable challenging probes, such as those with high GC and AT content, to be appropriately represented in the panel. The individual probes are synthesized on a larger scale than that which is available for array manufacturing, allowing IDT to produce a single, large lot of material that is aliquoted over time, providing consistent results for years.

xGen Exome Research Panel v2 consists of 415,115 individually synthesized and quality controlled xGen Lockdown Probes. The panel spans a 34 Mb target region (19,433 genes) of the human genome and covers 39 Mb of probe space, the genomic regions covered by probes. Probes were designed using our new “capture-aware” algorithm and assessed with proprietary off-target analysis, ensuring the most complete design coverage. All probes in the panel are manufactured under strict ISO 13485 standards. Mass spectrometry and dual quantification measurements of each probe ensure the quality of the probe and its appropriate representation in the pool.

Performance

Consistent data—don't waste time troubleshooting lots

Our lots last years not months.

Exome_Figure1

Figure 1. Limit expensive revalidation by having a single, large lot. 100 ng DNA was used to make libraries, which were captured in 8-plex. Two different users performed the captures on different days in different locations. The IDT xGen Exome Research Panel v2 shows a linear regression line that mimics the predicted “perfect” correlation line with an R2 value of 0.76. In comparison, 2 lots of supplier X showed significantly less consistency and indicate more lot-to-lot variation, which may require more detailed re-validation.

Superior on-target coverage and uniformity

xGen Exome Research Panel v2 reduces sequencing costs

Figure 2. The IDT xGen Exome Research Panel v2 outperforms other vendors for efficiency of capture. Sequencing libraries were prepared using enzymatic shearing and ligation-based library preparation using the library prep from each vendor. Libraries were captured and multiplexed according to the vendor’s exome panel capture protocol (X-8, R-8, and A-8, where 8 signifies 8-plex captures). For IDT, we show both 1-plex and 12-plex captures (IDT-1 and IDT-12, respectively). Enriched libraries were sequenced on a NextSeq® instrument (Illumina) in high output mode using 2x100 paired-end reads. On-target bases were determined with Picard (percent selected bases) using 5 Gb per library.

Highly uniform sequence coverage with xGen Exome Research Panel v2 leads to lower sequencing costs.

Figure 3. Highly uniform sequence coverage with xGen Exome Research Panel v2 leads to lower sequencing costs. DNA libraries were created from 100 ng of human genomic DNA (Coriell) using xGen Stubby Adapter and Unique Dual Index Primer Pairs with the Lotus DNA Library Prep Kit. These libraries were enriched either as 1-plex captures or in a single 12-plex capture using the xGen Exome Research Panel v2. The enriched libraries were sequenced (2x100) on a NextSeq® instrument (Illumina) and subsampled to 5 Gb. The data shows deep, uniform coverage with a flanked on-target rate of 94.7%, mean target coverage of 64.5X, and a duplication rate of 3.3% (calculated with Picard).

Excellent coverage of RefSeq database with minimal sequencing

Coverage profile of xGen Exome Research Panel v2 most closely resembles whole genome data.

Figure 4. Coverage profile of xGen Exome Research Panel v2 most closely resembles whole genome data. (A) A comparison of coverage depths of different exome panels to coverage depth from whole genome sequencing (WGS) of a matched library shows that the xGen Exome Research Panel v2 provides the closest match to WGS. Analysis of GC content shows that the higher coverage of the xGen panel is driven by the more effective capture of GC content. (B) RB1 exons 1 and 2 show extremes of GC content with ~76% in exon 1 and ~38% in exon 2. A comparison of capture between IDT and supplier A shows uniform, complete coverage for xGen Exome Research Panel v2 on the Integrative Genomics Viewer (Broad Institute), whereas supplier A struggles with GC content differences.

The most complete exome coverage is achieved with the xGen Exome Research Panel v2

Figure 5. The most complete exome coverage is achieved with the xGen Exome Research Panel v2. (A) Enriched libraries were sequenced with 5 Gb per samples and the percent of exons covered end-to-end at each read depth were calculated. xGen Exome Research Panel v2 (IDT) shows the highest percentage of exons covered at each indicated depth (compared to suppliers X, R, and A). (B) The xGen Exome Research Panel v2 provides the most complete end-to-end gene coverage at ≥20X. Individual samples were subsampled at different read depths (2x100 bp read length). The percentage of genes that were covered for every base of every exon at ≥20X was calculated at each read depth and plotted.

Maximize sequencing efficiency

xGen Exome Research Panel v2 reduces sequencing costs

Figure 6. xGen Exome Research Panel v2 reduces sequencing costs. DNA libraries were created from 100 ng of human genomic DNA (Coriell) using xGen Stubby Adapter and Unique Dual Index I Primer Pairs with the Lotus DNA Library Prep Kit. These libraries were enriched either as 8-plex (suppliers X, R, and A) or 12-plex (IDT) captures. The enriched libraries were sequenced (2x100) on a NextSeq® instrument (Illumina) and the number of reads required to achieve 75X mean target coverage (Picard) per sample was calculated. The number of samples that would fit on a NovaSeq™ S2 flowcell (Illumina) was calculated.

 

 

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