Genomic DNA was extracted from 500 μL of peripheral blood using a TIANamp Blood DNA Midi Kit (Tiangen, Beijing, China). After genomic polymerase chain reaction (PCR) performed, we sequenced the coding exons and their flanking intronic sequences of six crystallin family genes CRYAA, CRYBA1, CRYBB1, CRYBB2, CRYGC and CRYGD, for pathogenic mutations in the proband. The primers and reaction conditions used in PCR have been described in earlier reports [16 (link)–21 (link)]. To more clearly show the mutation c.451_452insGACT in CRYGD, the obtained fragment was cloned into pMD-18T vector (TaKaRa, Dalian, China) and sequenced. To confirm the mutation, we performed genomic polymerase chain reaction (PCR) using primers CRYGD forward (5’-TACGAGCTGTCCAACTACCGAG-3’ ) and reverse (5’-GAGAAATCTATGACTCTCCTCAG-3’ ) in the family and 103 unrelated individuals. The PCR fragments were separated by 8% polyacrylamide gel electrophoresis. The analysis of amino acid conservation around the mutation site was carried out by CLC DNA Workbench.
Tianamp blood dna midi kit
Manufactured by Tiangen Biotech
Sourced in China
The TIANamp Blood DNA Midi Kit is a laboratory equipment product designed for the extraction and purification of genomic DNA from whole blood samples. It utilizes a silica-based membrane technology to efficiently capture and purify DNA, providing a reliable and consistent method for DNA isolation.
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Lab products found in correlation
Novaseq 6000 platform, by Illumina (2 mentions)
Pmd18 t vector, by Takara Bio (1 mentions)
Tianamp bacteria dna kit, by Tiangen Biotech (1 mentions)
Abi 3730xl instrument, by Thermo Fisher Scientific (1 mentions)
Sequencer software, by Gene Codes (1 mentions)
Ion torrent pgm, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna blood midi kit, by Qiagen (1 mentions)
Nanodrop 8000, by Thermo Fisher Scientific (1 mentions)
Fastqc, by Babraham Bioinformatics (1 mentions)
Agilent sureselect human all exon v6 kit, by Agilent Technologies (1 mentions)
Qiaamp dna micro kit, by Qiagen (1 mentions)
Clc dna workbench, by Qiagen (1 mentions)
Taq pcr master mix, by Agilent Technologies (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Xgen exome research panel v1, by Integrated DNA Technologies (1 mentions)
Telmisartan, by LGC (1 mentions)
Nadph regenerating system, by Promega (1 mentions)
Supersignal west pico trial kit, by Thermo Fisher Scientific (1 mentions)
Primestar hs dna polymerase and restriction enzymes, by Takara Bio (1 mentions)
4 hydroxydiclofenac, by LGC (1 mentions)
13 protocols using tianamp blood dna midi kit
1
Genetic Screening for Crystallin Mutations
2
Extraction and Purification of gDNA from Whole Blood and Plasma
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Human gDNA samples were extracted from 0.5–2 mL of human whole-blood samples by using the TIANamp Blood DNA Midi Kit (DP332; Tiangen Biotech, Beijing, China) and eluted in 50 μL. The total DNA was isolated from 2 mL of the platelet-rich plasma spiked with bacterial species by using the same kit and eluted in 50 μL; bacterial gDNA was purified using the TIANamp Bacteria DNA Kit (DP302; Tiangen Biotech) and eluted in 50 μL. All procedures were performed in accordance with the manufacturer’s instructions. The concentration and purity of the isolated nucleic acids were determined using a NanoDrop 2000 spectrophotometer.
3
Validating Variants Using Sanger Sequencing
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The remaining variants identified from the WES analysis were further validated using Sanger sequencing. Genomic DNA was extracted from probands with suspected variants and their family members using a TIANamp Blood DNA Midi Kit (Tiangen Biotech Co., Beijing, China) according to the manufacturer’s instructions. The variants were amplified from genomic DNA by polymerase chain reaction (PCR) using gene-specific primers. The PCR amplification was performed using a TechNet Genius Thermo Cycler (TechNet Inc., Princeton, NJ, USA) and the following cycling program: initial denaturation at 95 °C for 10 min; 35 cycles of denaturation at 95 °C for 30 s, annealing for 30 s (annealing temperatures are listed in File S1 ), and extension at 72 °C for 30 s; and a final extension at 72 °C for 5 min. The resulting PCR products were sequenced using an ABI3730XL instrument (Applied Biosystems) and the DNA sequences were compared using the Sequencer software (Gene Codes Corp., Ann Arbor, MI, USA).
4
Genetic Diagnostic Sequencing for Inherited Retinal Dystrophy
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Genomic DNA was analyzed with targeted panel sequencing (each of six panels containing 70, 316, 78, 370, 429, and 386 genes) or whole exome sequencing (WES). Genes included in the panels are listed in Text S1 ; these genes are primarily responsible for inherited retinal dystrophy. Genomic DNA was isolated from leukocytes of venous blood samples using the QIAamp DNA Blood Midi Kit (Qiagen) or TIANamp Blood DNA Midi Kit (TIANGEN Biotech), in accordance with the manufacturer's standard protocol. Library preparation was performed using the Ion AmpliseqTM Library Kit 2 or SureSelect Exome V5 Capture library, in accordance with the manufacturer's instructions (Biswas et al., 2017 ; Chen et al., 2013 ; Javadiyan et al., 2018 ). Sequencing was performed on an Ion Torrent PGM (Life Technologies) or HiSeq (Illumina) platform.
5
Genomic DNA Sequencing and Analysis
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Genomic DNA was isolated using the TIANamp Blood DNA Midi Kit (TIANGEN Biotech, Beijing China) and fragmented to 150 bp using an ultrasonoscope (Covaris S220, Massachusetts, USA). End repair, adenylation and adapter ligation were performed for library preparation using a standard library construction kit (MyGenostics Inc., Beijing, China). Targeted DNA fragments were captured by a sequence capture array (MyGenostics Inc., Beijing, China). High-throughput sequencing and processing and bioinformatic data analysis were performed using the DNBSEQ-T7 sequencing platform (MGI Tech Co, Shenzhen China). The raw sequence reads were filtered using the BWA MultiVision software package and then aligned to GRCh38/hg38 (University of California Santa Cruz version). SNPs and indels were identified using the GATK Indel Genotyper and ANNOVA software. A CNV analysis was performed using the log2 ratio of the read depth on each exon.
6
Whole-Exome Sequencing of Genetic Variants
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With written consent, patients’ DNA was extracted from 5 ml peripheral blood using the TIANamp Blood DNA Midi Kit (Tiangen Biotech, Beijing, China). The quantity of DNA samples and the purity of nucleic acids were detected on a Nanodrop 8000 (Thermo Scientific, Waltham, MA, USA). A total of 57 probands and 364 controls were sequenced by WES. Whole-exome capture was performed by in-solution hybridization using a Biorupter to acquire 150–200-bp fragments. High-throughput sequencing was performed by massively parallel sequencing reads on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA). The analysing software FastQC (Babraham Bioinformatics, Cambridge, UK) was used to remove low-quality reads. The resulting reads were mapped to the human genome reference hg19 by Burrows-Wheeler Aligner. The Genome Analysis Toolkit (GATK Best Practices (Broad Institute, Cambridge, MA, USA)) was used for debugging base quality scores, realigning indels, and removing duplicates. The GATK VariantRecalibrator and ApplyRecalibration commands with the parameter ‘’--ts_filter_level 99.0.’’ were used to recalibrate variant scores.
7
Comprehensive Genomic Profiling of Tumor Samples
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Genomic DNA (gDNA) from peripheral blood was extracted by TIANamp Blood DNA Midi Kit (TIANGEN, China). gDNA from the matched tumor samples was extracted by QIAamp DNA Micro Kit (Qiagen, Germany). After DNA qualification, the sequencing libraries were constructed by Agilent SureSelect Human All Exon V6 kit (Agilent Technologies, USA). Sequencing was done by Illumina Novaseq 6000 platform (Illumina Inc., San Diego, USA) in Novogene Bioinformatics Technology Co., Ltd (Beijing, China). Finally, the ≈150 bp paired‐end reads were generated with a minimum coverage of 10× for ≈99% of the human genome (mean coverage was 100× for gDNA from peripheral blood, and 150× for gDNA from frozen tumor sample).
The somatic mutations were called by Mutect2 following GATK's best practice pipeline (https://gatk.broadinstitute.org/hc/en‐us/articles/360035894731 ). FASTQ data were aligned to human genome reference (hg19_b37) by Burrows‐Wheeler Aligner (BWA),[48 ] followed by marking duplicates, Base Quality Score Recalibration. The candidate variants were called and filtered through estimated Contamination and Orientation Bias. Somatic mutations passing the filters were then annotated by funcotator.
The somatic mutations were called by Mutect2 following GATK's best practice pipeline (
8
Identifying LDLR Pathogenic Mutations
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We extracted genomic DNA from 500 μL of peripheral blood using a TIANamp Blood DNA Midi Kit (Tiangen, Beijing, China). After performing genomic polymerase chain reaction (PCR), we sequenced the coding exons and their flanking intronic sequences of LDLR (GenBank NM_000527.4) for pathogenic mutations in the family members. The primers used in PCR have been described in an earlier report [13 ]. We screened for mutations in LDLR by direct sequencing. To verify the mutation, we separated heterozygous alleles by cloning the affected fragment into an EGFP-N1 vector. The fragment was amplified by PCR using forward primer 5′-TGAAATCTCGATGGAGTGGGTCCCATC-3′ and reverse primer 5′-CTGTAGCTAGACCAAAATCACCTATTTTTACTG-3′ and then cloned into EGFP-N1 vector. Plasmids were extracted from colonies and sequenced using the same primer. To confirm the novel mutation in LDLR, we also examined the mutation in the 100 unrelated controls. We performed the analysis of amino acid conservation around the mutation site using a CLC DNA Workbench (QIAGEN Bioinformatics, Germany).
9
Verifying WES Results via Sanger Sequencing
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To verify the WES results, the related EDA and WNT10A fragments were sequenced using Sanger sequencing. Genomic DNA from the brothers was isolated according to the procedure using a the TIANamp Blood DNA Midi Kit (Tiangen, Beijing, China) according to the manufacturer’s procedure. The primers used were specifically designed to detect the variations (Table 1 ). The coding sequences of the EDA and WNT10A genes were amplified using PCR with Taq PCR Master Mix (BioTek, Beijing, China). The PCR products were sequenced by Tsingke Biotechnology Co., Ltd. (Beijing). The results were compared with the reference sequences for each gene (EDA, NM_001399; WNT10A, NM_025216) (UCSC, http://genome.ucsc.edu/ ) to verify the results of WES.
Gene variations and primer sequences
| Variation | Forward primer | Reverse primer |
|---|---|---|
| EDA c.878 T > G (p.L293R) | 5′-AAGTTTGGCCTTCTAGGCTACC-3′ | 5′-CCTGCACCGGATCTGCATTC-3′ |
| WNT10A c.511C > T (p.R171C) | 5′-CGCTTTTGCCTACGCCATC-3′ | 5′-AACTCGGTTGTTGTGAAGCC-3′ |
| WNT10A c.637G > A (p.G213S) | 5′-CGCTTTTGCCTACGCCATC-3′ | 5′-AACTCGGTTGTTGTGAAGCC-3′ |
10
Maternal GR-1F Promoter Methylation
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Venous blood samples collected from 30 mothers were used to assess the methylation of the GR-1F promoter of the NR3C1 exon. Genomic DNA was extracted using the TIANamp Blood DNA Midi Kit (Tiangen, China). Quantification of purified DNA was carried out using an ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). Samples of DNA (500 ng) were bisulfated using the QIAGEN DNA methylation kit (QIAGEN, Valencia, CA, USA) and stored at −80°C.
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