High-Throughput Sequencing Library Preparation Solution

High-Throughput Sequencing is a revolutionary DNA/RNA sequencing technique that allows simultaneous sequencing of a large number of nucleotides in a short time frame. Through streamlined library preparation, sequencing, and data analysis steps,high-throughput sequencing enables comprehensive analysis of genomes, transcriptomes, and epigenomes. With higher throughput, faster speeds, and lower costs than conventional Sanger sequencing, high-throughput sequencing has become an essential tool for scientific research.

Library quality directly impacts sequencing performance, making library preparation the most critical step in the entire workflow. Library preparation typically involves DNA fragmentation, end-repair, dA-tailing, adapter ligation, PCR amplification, and purification, with enzymes playing a vital role throughout these processes. We will introduce the key enzymes, reagents, kits, and other services that we provide for library preparation.

Library Construction

The first step in library preparation is DNA fragmentation to obtain DNA molecules of adequate size. The two primary approaches are enzymatic fragmentation and sonication. Enzymatic fragmentation uses restriction endonucleases that specifically recognize and cleave phosphodiester bonds between phosphate and deoxyribose. By controlling the enzyme amount and digestion time, multiple samples can be processed simultaneously. However, this approach exhibits sequence bias and requires high-quality input DNA. Alternatively, dual-activity transposases can be utilized to fragment genomic DNA while adding specific adapters to the fragment ends. This combines DNA fragmentation, end-repair, and adapter ligation into a single step, effectively streamlining the workflow.

DNA Fragmentation

Random fragmentation of double-stranded DNA can produce a variety of termini, including 3' and 5' overhangs and 3'-phosphorylated and 5'-dephosphorylated ends. The most common configurations are sticky ends and blunt ends.

End Repair/dA-Tailing

T4 Polynucleotide Kinase (T4 PNK), derived from T4 bacteriophage-infected E. coli, catalyzes ATP γ-phosphate transfer to the 5'-hydroxyl terminus of double- or single-stranded DNA and RNA, as well as hydrolysis of 3'-phosphate groups from the 3' termini of oligonucleotides. It can be used for 5'-phosphorylation and 3'-dephosphorylation of DNA/RNA for ligation; and DNA/RNA end-labeling for probes and DNA sequencing.

Since many ligases have affinity for adenine (A) residues, As are often added to DNA ends to improve ligation efficiency. A-tailing can be performed using Klenow Fragment (3'→5' exo-).

Adapter ligation is the process of joining A-tailed DNA fragments to T-tailed adapters using T4 DNA ligase. Adapters are short DNA sequences that add indices to the library and attach target DNA fragments to the sequencer's flow cell. In addition to ligating adapters to the ends of DNA fragments, some protocols incorporate unique indices for individual samples, allowing multiplexed sequencing of pooled libraries.

Adapter Ligation

In high-throughput sequencing, having too many or too few input library molecules can compromise data quality. Therefore, quantitative analysis and qualitative evaluation of the sequencing library are critical for generating high-quality sequencing data. After PCR enrichment, library DNA quantity and purity are usually verified to quantify the number and size distribution of inserts and determine the appropriate library input for sequencing or downstream applications such as exome enrichment.

Fueled by powerful high-throughput core technologies combined with our synthetic biology, protein engineering, and biochemistry expertise, GCATbio offers a diverse portfolio of custom reagents spanning the entire high-throughput sequencing workflow. We also provide key products and end-to-end solutions tailored to your unique needs, available in easy-to-use formats.