차세대 염기서열분석 (Next generation sequencing, 이후 NGS)은 이전 시퀀싱 방법에 비해 현저히 빠르고 비용 효과적이며, 고효율 핵산 시퀀싱 기술을 의미합니다. 이 기술은 분자 생물학 및 유전체 연구에 혁명을 일으켰으며 개인화 된 의학의 미래에 대한 큰 기대를 가지고 있습니다.
Thermo Scientific KingFisher 자동 핵산 추출장비는 마그네틱 입자 기술을 사용하여 생체 분자를 정제합니다. 기기 기능은 추출 프로토콜에 필요한 일련의 솔루션을 통해 마그네틱 비드 바인딩 샘플을 이동시키는 것을 기반으로 합니다. KingFishers 장비는 다양한 NGS 샘플 준비 단계에 매우 적합합니다. 비드 - 샘플 복합체 만이 옮겨지기 때문에, 잔류 세척 버퍼 또는 다른 불순물의 양은 최소화됩니다.
사용하기 쉽고 직관적인 BindIt 소프트웨어가 제공되는 KingFisher 기성 프로토콜은 사용자가 새 프로토콜을 쉽고 빠르게 설정하거나 기존 스크립트를 수정할 수 있게 합니다.
KingFisher Sample Purification System
Next generation sequencing sample preparation on the KingFisher platform
Authors: Janica Djupsjöbacka and Pertteli Salmenperä, Blueprint Genetics, Helsinki, Finland
Maria Voutilainen, SP&A Application Laboratory, Thermo Fisher Scientific, Vantaa, Finland
At present, several alternative platforms exist for NGS, yet the basic sample preparation is uniform and contains the following steps:
1. Nucleic acid extraction (DNA or RNA)
2. DNA fragmentation
3. Adapter sequence ligation
4. Size selection of the fragments
5. PCR amplification (optional)
6. Quantification of the obtained library
Despite the advances in sequencing technology, manual NGS sample preparation is still labor intensive and time consuming, and includes numerous purification steps. The many benefits of automating the sample purification include reduced hands-on work and improved consistency between the processed specimens, all of which translates into enhanced laboratory output.
NGS sample preparation workflow assisted by the KingFisher instrument, presented in Figure 1, is based on the methodology employed by Blueprint Genetics, a molecular genetics laboratory that provides genetic diagnostics and clinical interpretation for hereditary diseases.
NGS sample preparation begins with nucleic acid extraction from the studied specimen. The extraction can be done either manually or with various automated instruments, including the KingFishers. At Blueprint Genetics, genomic DNA is isolated from whole blood or saliva. The obtained DNA is fragmented into smaller pieces mechanically using a focused-ultrasonicator (Covaris). The desired sized fragments are then isolated on a KingFisher Duo Prime with magnetic beads (Beckman AMPure SPRIselect). Uneven single-stranded ends of DNA (sticky ends) present on the fragments are repaired concurrently (NEBNext® Ultra™ DNA Library Prep Kit, New England BioLabs).
Figure 1. NGS sample preparation workflow.
1) Genomic DNA extraction from blood or saliva.
2) DNA fragmentation.
3) Selection of correct sized fragments with magnetic beads on the KingFisher instrument.
4) End repair and adaptor ligation to 5’ and 3’ ends of the fragments.
5) Sample purification on a KingFisher instrument to remove unused adapters.
6) PCR amplification of the sample.
7) Post-PCR purification on a KingFisher instrument.
8) Sample pooling, sequencing and analysis.
Specific adapters—short strands of nucleotides with known sequences and specialized barcode portions— are next ligated to both ends of the fragments. Any leftover adapters are removed in the following clean-up steps on the KingFisher Duo Prime. Library enrichment refers to PCR amplification of the samples and is performed to ensure adequate DNA yield for the sequencing. The last purification step removes unligated adapters, possible adapter-dimers and unused primers, as well as additional PCR reaction components. The step is carried out with a dedicated post-PCR KingFisher Duo Prime to guarantee that no amplicon contaminants are spread.
Finally, the samples are pooled and sequenced. The barcode sequences allow for multiple sample analyses on a single run. DNA quality control of fragment size, purity and concentration is performed several times during the process (Figure 1). At Blueprint Genetics the sequencing is accomplished by applying a targeted sequencing approach called Oligonucleotide-Selective Sequencing (OS-Seq™), (Myllykangas et al., 2011). High-quality sequencing data from the clinically significant target genes is analyzed on Blueprint Genetics’ automated bioinformatics pipeline, and a team of geneticists and clinicians interprets the results to prepare a clinical statement.
KingFisher instruments are like a reliable extra set of
hands for several NGS sample preparation steps. When purity and minimal
sample-to-sample variation are requisite, automation with the KingFishers helps
to procure top-grade samples with less hands-on time.
Myllykangas, Buenrostro, Natsoulis, Bell and Ji. Efficient targeted resequencing of human germline and cancer genomes by oligonucleotide-selective sequencing. Nature Biotechnology 2011;11:1024-7.
NGS work flow including sample purification with the KingFisher Duo Prime
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