The PCR-free NGS DNA Library Prep Kit was developed for construction of DNA libraries for next generation sequencing (illumina platform). The kit adds 3′-dT-tailed library adapters to both ends of DNA fragments efficiently. The kit uses double strand DNA fragments (blunt and/or sticky) as input DNA for NGS library construction, and is compatible with DNA fragments generated from both enzymatic methods (BioDynami DNA fragmentation enzymes etc.) and physical methods (sonication, nebulization etc.).

PCR-Free NGS DNA Library Prep Kit Workflow

PCR amplification is a standard step for library preparation of Next-Generation Sequencing. The PCR is used to amplify fully ligated DNA fragments and to add index information to the libraries. The indexing is for the pooling of the library samples in an effort to reduce the sequencing cost.

However, PCR introduces uneven amplification of DNA in some DNA regions with extreme GC-contents and secondary structures. This bias can cause very low sequencing coverage in such regions. DNA sequencing in these regions is still a huge challenge.

PCR-free library prep can reduce library bias and minimize sequencing gaps. The sequencing data from PCR-free library samples have even genomic coverage with few gaps and better depth in GC-rich regions. Our PCR-free NGS kit offers optimal coverage in the regions that are traditionally difficult, such as high-GC content regions, low-GC content regions, and repetitive sequence regions.

Two index types are available for the kit:

Non-index: Libraries do not have index.

Index: Each library contains one i5 index and one i7 index.  Library multiplexing up to 96 samples is possible. List of indexes can be downloaded Here.

Kit advantages:

• • • Total time: 1 hr
    • Hands-on time: 5 min
    • Easy procedure: Ready-to-use master mix & Less reaction components
    • Input DNA amount from 100 ng to 1 ug

The PCR-free NGS DNA Library Prep Kit was developed for construction of DNA libraries for next generation sequencing (illumina platform). The kit adds 3′-dT-tailed library adapters to both ends of DNA fragments efficiently. The kit uses double strand DNA fragments (blunt and/or sticky) as input DNA for NGS library construction, and is compatible with DNA fragments generated from both enzymatic methods (BioDynami DNA fragmentation enzymes etc.) and physical methods (sonication, nebulization etc.).

Overview

• Protocol optimized for DNA isolated from a diversity of samples including stool, soil, water, saliva, plant, urine, skin, and more
• Simple and quick workflow: library could be prepared in less than 5 hours
• Component of Norgen’s metagenomics workflow
• A single NGS run can be prepared with up to 384 unique dual-index libraries

Sample type purification kit guide 

The 16S V2-V3 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V2-V3 amplicon libraries to be used for next-generation sequencing on Illumina platforms. All molecular reagents including primers, enzyme mixes, indexes, and buffers are provided. Instructions for PCR clean up with the AMPure XP Magnetic Beads (supplied by customer) are also included for rapid purification of nucleic acid products generated at two steps of the workflow. The library prep workflow could be used for purified DNA inputs from different sources including stool, soil, water, saliva, plant, urine, skin swab, vaginal swab, cheek swab, nasal swab, plasma/serum, tongue swab, gum swab, and others.

The 16S V2-V3 Library Preparation Kit for Illumina has a streamlined procedure that reduces the handling time such that the library prep procedure can be completed in approximately 4 hours (see diagram below). Input DNA is first subjected to targeted PCR to amplify the V2-V3 region of the DNA encoding 16S rRNA. The post-PCR reaction is then cleaned up using AMPure XP beads. Dual index primers are then added using a limited-cycle PCR. The indexed amplicons flanked by 5′ and 3′ barcoded adaptors are then cleaned using AMPure XP beads. The libraries are then ready for quantification, pooling and sequencing.

Details

Supporting Data

Figure 1 / 3

Previous

Next

Click for expanded view

Storage Conditions and Product Stability
Norgen’s 16S V2-V3 Library Prep Kit for Illumina is shipped as one kit box (for the 24 prep kit) or two sub-component kits (for the 96 prep kit). All kits should be stored at -20°C upon arrival.

All kit components should remain stable for at least 1 year when stored at the specified storage conditions.

endo-BCN-PEG2-PFP ester is a click chemistry active ester. The PFP esters have similar applications as the NHS esters, but are more stable in aqueous solution. They can be used to label the primary amines (-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The BCN group can react with azide-tagged biomolecules. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.

endo-BCN-PEG2-PFP ester is a click chemistry active ester. The PFP esters have similar applications as the NHS esters, but are more stable in aqueous solution. They can be used to label the primary amines (-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The BCN group can react with azide-tagged biomolecules. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.