Ngs Library Prep

[Andrew Schiavo]

NGSlibrary preparation with continual innovation. The result: a workflow that is even easier to use, scalable for any size lab, requires a small number of steps, and has a fast workflow time. The library preparation process involves converting a genomic DNA sample (or cDNA sample) into a library of fragments which can then be sequenced on an NGS instrument. Breakthrough technology in our library prep helps you get to answers in less time.

Illumina library prep protocols accommodate a range of throughput needs, from lower-throughput protocols for small labs to fully automated library preparation workstations for large laboratories or genome centers. Our solutions support a broad range of sample types, from cell lines to fresh tissue, formalin-fixed paraffin-embedded (FFPE) samples, blood, and other challenging sample types.

At Illumina, our goal is to apply innovative technologies to the analysis of genetic variation and function, making studies possible that were not even imaginable just a few years ago. It is mission critical for us to deliver innovative, flexible, and scalable solutions to meet the needs of our customers. As a global company that places high value on collaborative interactions, rapid delivery of solutions, and providing the highest level of quality, we strive to meet this challenge. Illumina innovative sequencing and array technologies are fueling groundbreaking advancements in life science research, translational and consumer genomics, and molecular diagnostics.

Included are all necessary reagents to perform end repair, adaptor ligation, combinatorial dual indexing for multiplexing up to 48 samples, and DNA cleanup with SPRIselect reagent from Beckman Coulter, Inc. (see Additional Information) If additional multiplexing is desired, Primer Sets 1 and 2 can be combined for sequencing of up to 96 samples in a single lane. Pairing this kit with the EpiCypher CUT&RUN Kit (EpiCypher 14-1048) affords users a cells-to-sequencing solution for chromatin mapping experiments with all the necessary controls and validated reagents to ensure confidence in obtaining high quality data. To place bulk orders, contact in...@epicypher.com.

Figure 1: High quality sequencing libraries from as little as 0.5 ng of input

Gene browser shots generated using the Integrative Genomics Viewer (IGV, Broad Institute) show a representative 137 kb window for H3K27me3 antibody (ABclonal A16199) tested in CUT&RUN using 500k K562 cells. Decreasing inputs of CUT&RUN enriched DNA were used in the library prep protocol to simulate low abundance targets or low cell input experiments. Data are largely indistinguishable across inputs, demonstrating robust preparation of Illumina NGS-ready libraries.

This Product is covered by one or more patents, trademarks and/or copyrights owned or controlled by NEB. While NEB develops and validates its products for various applications, the use of this product may require the purchaser to obtain additional third-party intellectual property rights for certain applications.

The authors investigated the microbial pools associated with landfill reactors that are impacted by nanomaterials. By preparing 16S rRNA libraries with the Quick-16S NGS Library Prep Kit, they discovered that the bacterial communities were similar between control reactors and nanomaterial-containing reactors, but the archaea communities were highly different.

This study investigated how varying interchange ratios in wastewater treatment affects microbial community composition. Analysis of samples prepared for 16S sequencing with the Quick-16S NGS Library Prep Kit demonstrated that the relative abundance of the phyla Proteobacteria, Acidobacteria, and Bacteroidetes varied according to the interchange ratio and that the species of Thiothrix were highly abundance in all samples, revealing their potential importance to wastewater treatment processes.

Akyol et al. performed 16S and 18S analysis of anaerobic digestates using the Quick-16S NGS Library Prep Kit to assess microbial diversity in lignocellulose-rich digestates. The most abundant bacterial and fungal genera were consistent across digestate samples.

This study is the first to investigate the bacterial community of the egg parasitoid Telenomus tridentatus. Using libraries prepared with the Quick-16S NGS Library Prep Kit, Ramrez-Ahuja et al. showed the gut bacterial community of this parasitoid wasp is dominated by Proteobacteria and Actinobacteria and lay the groundwork for future studies regarding the role of bacterial communities in wasp species.

Improving the methane production and energy yield of biogas reactors by fungal bioaugmentation was investigated in this research. Results from testing with the Quick-16S NGS Library Prep Kit for 16S rRNA analysis revealed that both fungal bioaugmentation and the different use of either whole crop or crop residues influenced the composition of the bacterial community in the reactors, with several taxa only identified in the bioaugmented samples, but that neither factor influence the methanogenic archaea composition, even though the methane yield in bioaugmented samples increased by 15-33%.

Illumina DNA Prep, previously known as Nextera DNA Flex, offers a fast, robust, and flexible workflow for preparing normalized, sequencing-ready libraries from a wide range of DNA input types and amounts facilitating an array of applications, from human whole-genome sequencing to sequencing amplicons, plasmids, and microbial species.1

Illumina DNA Prep workflow delivers consistent insert sizes, uniform coverage, and optimized performance, regardless of DNA input amount or genome size. The bead-based technology minimizes bias and opportunities for error, resulting in highly reproducible sequencing data.

This product is also available as an Illumina Advantage (TG) product. Illumina Advantage large-scale sequencing products feature lot-specific shipments and testing, extended shelf life, and advanced change notifications for greater laboratory efficiency.

(A) Bead-linked transposomes mediate the simultaneous fragmentation of gDNA and the addition of Illumina sequencing primers. (B) Reduced-cycle PCR amplification amplifies sequencing ready DNA fragments and adds indexes and adapters. (C) Sequencing-ready fragments are washed and pooled.

Comparison of the total workflow time from DNA extraction to library normalization and pooling. Illumina DNA Prep produces a workflow with the least number of steps and the fastest total workflow time.

(A) Illumina DNA Prep delivers uniform coverage across the genome comparable to the TruSeq DNA Nano kit. (B) Coverage is shown for microorganisms with extremely high- or low-GC content. Due to improved on-bead tagmentation library prep chemistry, Illumina DNA Prep shows more even coverage than Nextera XT.

Coverage of GC-rich regions can be impacted by the model, settings, and performance of the thermal cycler used. Illumina has validated the Bio-Rad DNA Engine Tetrad 2, the Bio-Rad S1000, the Bio-Rad C1000, and the MJ Research PTC-225 DNA Engine Tetrad thermal cyclers for use with Illumina DNA Prep. Other thermal cyclers may differ in their performance, which may impact genomic coverage.

In this 5-minute video, we walk you through the single-step workflow to demonstrate the speed and simplicity of the auto-normalizing ExpressPlex technology. The ExpressPlex Library Prep Kit is an ideal solution for high-throughput multiplexed sample preparation for plasmid and amplicon sequencing.

ExpressPlex demonstrates significantly higher levels of normalization compared to competitors, enabling a simplified workflow where individual normalization is no longer required to achieve more consistent read-depths across samples.

Libraries were prepared from three different reference plasmids of varying sizes across a 10-fold input range at full reaction volume for ExpressPlex (16 L) and Nextera XT (50 L), as well as at miniaturized reaction volume for Nextera XT.

Given the robust read balance and lower number of reads required to generate correct assemblies, ExpressPlex users can multiplex higher numbers of samples on a single Illumina run or utilize a lower capacity flow cell, thereby reducing the cost of sequencing per plasmid.

Rapidly prepare stranded, whole transcriptome sequencing libraries using Watchmaker RNA Library Prep Kits with Polaris Depletion. The highly streamlined Polaris Depletion module depletes highly abundant rRNA and globin transcripts in human, mouse, and rat samples, providing improved coverage of biologically interesting transcripts, including long non-coding RNAs.

The automation-friendly workflow reduces total turnaround time, hands-on time, and consumable use through a reduction in bead purification and reaction steps. A novel, engineered reverse transcriptase improves the conversion of RNA to cDNA, enabling high-quality performance with 1 ng to 1000 ng of total RNA, as well as FFPE-derived RNA.

Figure 1. Improve automatability and reduce turnaround time. The Watchmaker solution combines and shortens enzymatic steps and has fewer bead purifications in comparison to commercially-available kits, resulting in a highly automatable workflow with significantly reduced hands-on time (up to one hour per plate) and consumable use (up to 1,000 pipette tips per plate).

3a8082e126