Next-Generation Sequencing Library Preparation—Thermo Scientific

NGS Library Preparation Workflow

ClaSeek and MuSeek technologies

The Thermo Scientific product portfolio provides two distinct NGS library construction technologies – ClaSeek and MuSeek – each created to meet researcher's most demanding needs. The unmatched versatility of ClaSeek kits and incredible ease of use of MuSeek kits enables scientists to achieve excellent results with less effort and more time saved.

ClaSeek Library Preparation

Unmatched Flexibility
Thermo Scientific ClaSeek Library Preparation Kits are designed for fast, convenient and accurate DNA library construction from DNA sample input as low as 5 ng. Available amplification-free protocols eliminate any possible PCR-related bias, resulting in uniform data coverage and reliable sequencing results.

Features:

• PCR-free–significantly minimized bias and improved data coverage with amplification-free protocol
• Flexible–library construction from 5 ng to 1 µg DNA sample
• Fast and convenient–up to 50% shorter library preparation workflow with significantly less hands-on time

MuSeek Library Preparation

Speed and convenience
Thermo Scientific MuSeek library preparation kits utilize an innovative transposon-based technology that allows enzymatic fragmentation and simultaneous tagging of the target DNA, eliminating the need for physical shearing, DNA end-repair, and adapter ligation steps. This enables fast library construction with minimal hands-on time and less effort.

Features:

• Fast–library generation in less than 80 minutes
• Easy-to-use–no physical shearing, end-repair and ligation steps
• Universal–one protocol for generating different fragment length libraries

Enzymatic fragmentation and simultaneous tagging

MuSeek Library Preparation Kits utilize an innovative transposon-based technology that allows enzymatic fragmentation and simultaneous tagging of the target DNA, eliminating the need for physical sample shearing, DNA end-repair, and adaptor ligation steps.

The fragmentation and tagging reaction is catalyzed by a homotetrameric MuA transposase complex (75 kDa protein originating from bacteriophage Mu) containing two 50 bp double-stranded transposon DNA ends with a specific MuA binding sequence. Initially, the genomic DNA sample is simultaneously fragmented and tagged with transposon sequences at each end of resulting DNA fragments (1). In the next step the 3'-ends of the fragmented DNA are elongated over the 5 nt gaps generated during the transposition reaction and further extended into transposon sequences (2) . Sequencing platform-specific adaptors are added to the fragments in a subsequent PCR-based adaptor-addition reaction. In the initial cycles of PCR the first 16 nt of the adaptor 3'-ends hybridize to transposon sequences in tagged DNA fragments (2). In later PCR cycles the fragments are amplified using a pair of external primers.

Cleanup and Size Selection

Reliable NGS Library Cleanup and Size Selection
Thermo Scientific MagJET and GeneJET NGS Cleanup and Size Selection kits are optimized for robust and efficient DNA fragment library purification and size selection from a large variety of enzymatic reaction mixtures used in NGS workflows.

Features:

• Robust–reliable cleanup and size selection from different reaction mixtures and buffers
• Flexible–user-defined size-selection of exact DNA fragment range
• NGS-grade purity–complete removal of NGS adapters, primers, nucleotides, enzymes and other reaction components inhibiting NGS workflow
• High-throughput compatible–easy to automate on Thermo Scientific KingFisher magnetic particle processors for high-throughput sample handling

Robust cleanup and size selection

Fragmented E. coli genomic DNA (1 µg) samples, each spiked in different reaction mixtures and buffers (a-h) typical for next-generation sequencing workflow, were size selected using MagJET and other supplier protocols. Isolated fragment libraries were analyzed on an agarose gel. Expected size selection peak areas are marked with a dotted line.

a – End Conversion Reaction mixture, IT;
b – PCR Reaction mixture (1);
c – PCR Reaction mixture (2);
d – Ligation Mixture;
e – End Conversion Reaction mixture (IL);
f – Fragmentation reaction buffer;
g – Elution buffer;
h – Nebulization buffer;
C – Control (input DNA)

Efficient size selection of different fragment lengths