Solution|Hangzhou Allsheng Instruments

NGS Library Preparation

With the decline of sequencing costs and the continuous improvement of sequencing throughput, NGS technology has developed rapidly and its application scope has continued to expand. Molecular and genetic testing technologies are active in many fields such as early tumor screening, companion diagnosis, genetic diagnosis, infectious diseases, organ transplantation, pharmacogenomic testing, and non-invasive prenatal screening.

NGS technology enables high-throughput, high-depth detection of genomes in a very short period of time, making individualized and precise diagnosis and treatment possible. However, the experimental operation steps of NGS library preparation are cumbersome and lengthy, and even the most advanced laboratories are limited by the number of steps that are still performed manually, so that the experimenters are often confined to work for a long time in front of the test bench, which is not only the whole process is labor-intensive and time-consuming, but also prone to errors, resulting in waste of samples and reagents.

In order to solve the various problems in the above-mentioned NGS library preparation process, Allsheng has launched a full-process solution from nucleic acid extraction, library construction to hybrid capture. Among them, Auto-NGS series is an efficient, compact and unattended automatic NGS library preparation equipment. It helps to reduce the workload, process more samples in parallel, improve the repeatability of the library preparation process, greatly liberate laboratory manpower, minimize possible errors, and improve the stability of library preparation.

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NGS Library Preparation

Sample Pre-processing

The routine extraction of nucleic acid from animal and plant tissues often requires a process of sample crushing, and the tissue block needs to be liquefied to speed up the lysis of the sample. In addition, in the process of metagenomic sequencing, samples (such as bacteria, fungi, viruses, etc.) also need to be pretreated to better extract bacterial nucleic acid. Because conventional reagents are used to lyse the above-mentioned samples, the processing time is usually long and the effect is poor. Therefore, it is recommended that customers can use a biological sample homogenizer to grind the sample first to achieve the purpose of breaking the cell wall and accelerate the release of nucleic acids.

Instrument Features

· Up to 24×2 ml samples (Bioprep-24/24R) can be processed in one run
· Use a separate crushing tube for each sample, effectively avoiding cross-contamination
· Grinding beads are available in a variety of materials and diameters for different types of samples

Experimental Data

Nucleic Acid Extraction

Allsheng automatic nucleic acid extraction instrument series products meet the needs of different sample processing volumes and different sample processing volumes. The sample throughput is from 16-96, and the maximum sample processing volume is from 1mL-10mL. To meet the extraction requirements of blood genomic nucleic acid, cfDNA and FFPE sample nucleic acid, the open software design can be adapted to various magnetic bead extraction kits on the market.

Instrument Features

· Up to 24×10 mL reaction systems can be processed in one run, with a sample volume of 1-4 mL
· Mature reagent solution, supporting single reagent design, can be used for single sample extraction, convenient and flexible
· Commonly used for cfDNA

Experimental Data

Library Quality Control

In the process of library preparation, due to different sample sources and different characteristics of the samples, the same weight of samples and the same extraction method may result in different concentrations of nucleic acid. However, since some library preparation processes, such as adapter connection, sample homogenization, etc., require accurate nucleic acid concentrations as a basis, fluorometers have become the best choice in this process. Compared with micro-spectrophotometer, fluorometer detection is more specific; compared with PCR method, fluorescence detection operation is simpler.

Instrument Features

· Fluo-200/800 can detect 1/8 samples in one run
· High sensitivity - the lowest detection limit can reach 0.1pg/μL (dsDNA)
· Open system, in addition to standard reagents, optional reagents can also be matched

Experimental Data

Library Preparation

Auto-NGS 100 is a compact, flexible and easy to use library prep system. It is designed for efficient sample preparation of up to 24 NGS libraries. Auto-NGS 100 has a simple tabletop design and an intuitive user interface, which can avoid user misoperation. And it has a UV sterilization lamp and a high-efficiency purification filter device to avoid cross-contamination. Auto-NGS 100 adopts an intuitive GUI to guide users to select and edit the program to be run, quickly complete the layout of the table, and display the running status and detailed information in real time. The open design can accommodate different reagent suppliers and sequencing platforms.

Instrument Features

· High-precision pipetting, suitable for micro pipetting needs
· Open software design to match the needs of various library preparation kits in the market
· The software logic is clear, easy to use, and the learning cost is low
· Significantly shorten the library preparation time and standardize the process

Experimental Data

Sample Pre-processing
Nucleic Acid Extraction
Library Quality Control
Library Preparation

Sample Pre-processing
Nucleic Acid Extraction

Fragment analysis results of manual extraction and automated extraction of simulated samples (DNA ladder) (left) and actual extraction results of plasma from patients with different volumes of tumor (right)

Note: Fragment analysis was performed on 1 ul of the manually extracted sample and 1 ul of the automatically extracted sample at the same time. The results showed that the fragment distributions of the two were highly consistent, and the recovery efficiency was over 90% according to the quantitative results of the Fluo-100 fluorometer (Figure 2 left). The plasma of 0.5 ml, 1 ml and 4 ml of tumor patients was extracted by manual method and automated instrument respectively, and the elution volume was 50 ul. The results are shown on the right in Figure 2. There is no significant difference in the yields of the two extraction methods under the three volumes.
Library Quality Control

Fluo-200/800 common sample quantification range (left) and Allsheng® high sensitivity dsDNA quantification analysis kit with 0.2–100 ng linear quantification range (right)
Library Preparation