Blood samples were collected from individuals after obtaining the informed consent for this study. Genomic DNAs extraction was carried out using MagPure Buffy Coat DNA Midi KF Kit (Magen, China) according to the manufacturer's instruction. The quality and quantity of DNA samples were determined by NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). DNA samples with absorbance ratio A260/A280 between 1.6 and 2.1 and total amount greater than 2 μg were acceptable and used in following steps.
Magpure buffy coat dna midi kf kit
Manufactured by Magen Biotechnology Co
Sourced in China
The MagPure Buffy Coat DNA Midi KF Kit is a laboratory equipment designed for the automated extraction and purification of genomic DNA from buffy coat samples. It utilizes magnetic bead-based technology to efficiently capture, wash, and elute DNA, providing a reliable and consistent method for DNA isolation.
Automatically generated - may contain errors
9 protocols using magpure buffy coat dna midi kf kit
1
Genomic DNA extraction from blood
2
Library Preparation and Sequencing for Variant Discovery
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Library preparation and next-generation sequencing was performed at the Beijing Genomics Institute (BGI), China. Genomic DNA was extracted according to the manufacturer’s instructions from 300 μl of venous blood collected from the proband (III-9) using the MagPure Buffy Coat DNA Midi KF Kit (Magen, China). To generate short DNA fragments (100–500 bp), the extracted genomic DNA was fragmented using segmentase (BGI, China) and screened with magnetic beads to enrich the fragments with sizes ranging from 280 to 320 bp. Then, the ends were filled, an “adenine” base was added to the 3′ end to facilitate ligation of the DNA fragment to the adapter, and a “thymine” base was added at the 5′ end. A library was constructed using the purified DNA fragments amplified via a ligation-mediated polymerase chain reaction (PCR). The library was enriched by array hybridization, according to the manufacturer’s instructions (Roche NimbleGen, United States), eluted, and amplified post capture. The magnitude of product enrichment was estimated using an Agilent 2100 Bioanalyzer. All amplified libraries were subsequently sent to BGI for circularization and sequencing on the MGIseq-2000 platform using a paired-end 100 bp sequencing strategy. The sequencing reads were automatically demultiplexed using the index.
3
Whole Genome Sequencing of Proband and Parents
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Venous blood was withdrawn from the proband and his parents, and genomic DNA was extracted according to the manufacturer's standard procedure for the MagPure Buffy Coat DNA Midi KF Kit (Magen, Guangzhou, China). Then, the genomic DNA was fragmented by Segmentase (BGI, China) to generate small DNA fragments (100–500 bp) that were further screened using magnetic beads to enrich the fragments with sizes ranging from 280 to 320 bp. After the ends were filled, an “A” base was added to the 3' end to enable ligation of the DNA fragment to an adapter with a “T” base at the 3' end. The DNA fragments were amplified by a ligation-mediated polymerase chain reaction and purified to form a library. The library was enriched by array hybridization following a protocol (Roche NimbleGen, Madison, USA), followed by elution and postcapture amplification. The magnitude of the enrichment of the products was measured using an Agilent 2100 Bioanalyzer. All the amplified libraries were subsequently sent to BGI for circularization and sequencing on the MGIseq-2000 platform with a paired-end 100 sequencing strategy. The sequenced data were automatically demultiplexed by index.
4
Whole Exome Sequencing and Variant Analysis
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The whole exome sequencing and data analysis procedures were previously reported (5 (link)–7 (link)). Briefly, we first extracted genomic DNA from the blood samples according to the standard procedure of the manufacturer (MagPure Buffy Coat DNA Midi KF Kit, Magen, China), and the qualified genomic DNA was then sequenced with PE100 + 100 on MGISEQ-2000. We applied the BGI MGIEasy V4 chip, which contains exons of all human genes and their adjacent ± 20 bp introns, to capture the targeted sequences. Bioinformatics processing and data analysis were then performed to explore the potential variants after we received the primary sequencing data. Several databases, including the 1,000 Genomes Project, HapMap, NCBI dbSNP, and a database of 200 normal Chinese adults, were used to filter and estimate all the SNVs and indels. Finally, Sanger sequencing was used to validate all mutations and potential pathogenic variants. The Human Gene Mutation Database (HGMD)1 was introduced to screen previously reported mutations. To rule out the possibility of a polymorphism, the mutations were also blasted in the 1,000 Genomes Project,2 ExAC,3 HapMap,4 ESP6500,5 NCBI dbSNP,6 GnomAD,7 and a database of 200 normal Chinese adults.
5
SMN1 Exon 7 Copy Number Detection
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A total of 478 DNA samples tested previously with qPCR, MLPA or NGS for exon 7 copy number were enrolled in this study. They were collected from November 2014 to May 2019 from hospitals in mainland China. The informed consent was obtained from all subjects or, if subjects are under 18, from a parent and/or legal guardian. Sixteen samples were from SMA-diagnosed patients with SMN1 homozygous deletion, 74 samples were from the relatives of SAM patients with SMN1 heterozygous deletion, and the remaining 388 samples were from individuals without SMA-associated symptoms without family history information. Fifty-nine of the latter 388 samples were previously determined to have heterozygous exon 7 deletion of SMN1 by qPCR, MLPA or NGS. Genomic DNA was extracted from peripheral whole blood with the MagPure Buffy Coat DNA Midi KF Kit (MAGEN) following the manufacturer’s directions, and subsequently, the extracted DNA was used for the analysis of SMN1 exon 7 copy number with MLPA, qPCR and NGS simultaneously. This study was approved by the ethics committee of BGI (NO. BGI-IRB 18087).
6
CNV-seq Profiling and Classification
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For CNV-seq, DNA was extracted from peripheral blood using a MagPure Buffy Coat DNA Midi KF kit (Magen, Guangzhou, China), followed by DNA fragmentation, library construction using PCR technology, and index addition. The quality of the libraries was tested using a Qubit dsDNA HS Assay kit (Invitrogen, Waltham, MA, USA) before and after pooling the libraries, and the hybrid library was then subjected to 35 bp single-terminal sequencing with a sequencing depth of 0.41× using a DNBSEQ-G400 High-throughput Sequencing Set and the MGISEQ-2000 sequencer (MGI, Shenzhen, China). The results were determined by referring to the hg19 version of the human genome and the most recent data available on the Database of Genomic Variants, DECIPHER, Online Mendelian Inheritance in Man, University of California Santa Cruz, PubMed, ClinGen, DGV, and other public databases. Finally, the clinical significance of CNVs was divided into five classes according to the American College of Medical Genetics (ACMG) sequence variant classification guidelines for copy number variants [16 (link)]: pathogenic, likely pathogenic, benign, likely benign, and variant of uncertain significance (VUS).
7
Automated Genomic DNA Extraction
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Genomic DNA was extracted automatically from periphery whole blood with a MagPure Buffy Coat DNA Midi KF Kit (Magen, China). DNA sample quality and quantity were measured using gel electrophoresis and/or a NanoDrop spectrophotometer (Thermo Scientific, USA).
8
Whole Genome Sequencing Library Preparation
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Library preparation and next‐generation sequencing (NGS) were performed at the Beijing Genomics Institute (BGI), China. Genomic DNA was extracted from 300 μl of venous blood collected from the proband (III‐1) using the MagPure Buffy Coat DNA Midi KF Kit (Magen, China) according to the manufacturer's instructions. To generate short DNA fragments (100–500 bp), the extracted genomic DNA was fragmented using segmentase (BGI, China) and screened with magnetic beads to enrich the fragments with sizes ranging from 280 to 320 bp. Subsequently, the ends were filled and the base “Adenine” was added to the 3′ end to facilitate ligation of the DNA fragment to the adapter with a “Thymine” base at the 5′ end. A library was constructed using the purified DNA fragments amplified via a ligation‐mediated polymerase chain reaction (PCR). The library was enriched by array hybridization, according to the manufacturer's instructions (Roche NimbleGen, USA), eluted, and amplified postcapture. The magnitude of enrichment in products was estimated using an Agilent 2100 Bioanalyzer. All amplified libraries were subsequently sent to the BGI for circularization and sequencing on the MGIseq‐2000 platform using a paired‐end 100 bp sequencing strategy. The sequencing reads were automatically demultiplexed using the index.
9
DNA Extraction from Buffy Coat
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10 ml of whole blood was centrifuged at 3000 r/min for 10 min, and then 165 µl of buffy coat was obtained to extract DNA using MagPure Buffy Coat DNA Midi KF Kit (Magen, China). The DNA was sequenced on the BGISEQ-500 platform using 200 bp single-end reads. Data processing was performed using Immune IMonitor [60] (link).
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