Methods of preparing and analyzing nucleic acid libraries
A nucleic acid molecule and single nucleotide polymorphism technology, applied in the field of detection of single nucleotide polymorphisms and copy number variations in samples, can solve the problems of missing samples of clinical significance, mutations, etc.
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example 1
[0102] Example 1: Detecting Copy Number Variations (CNVs) and Single Nucleotide Polymorphisms (SNPs)
[0103] CNVs and SNPs are detected in the same sample using the present disclosure. Briefly, a pre-amplification procedure using WGA / WTA primers in combination with target-specific primers, followed by nested PCR assays using nested primers for targeted amplification, and indexed PCR for additive sequencing on a sequencer Desired sequences are determined to detect CNVs and SNPs in samples with a limited number of cells (eg, a single cell or five cells) or genomic DNA (eg, 30 pg of genomic DNA). Next-generation sequencing (NGS) assays were performed to generate sequence reads, which were analyzed by a custom bioinformatics pipeline to detect CNVs and SNPs. This method allows the detection of distinct mutations at a low sequencing depth of about one million reads.
[0104] Using with some modifications Gold Single Cell DNA-Seq Kit (Takara Bio USA, R300669) was used for det...
example 2
[0132] Example 2: Targeted amplification for SNP detection
[0133] In this example, we demonstrate the use of target-specific primers for the detection of SNPs, eg in carrier screening. 15 ng of genomic DNA (NA07552 or NA012785) was extracted from GM07552 or GM12785 cells, respectively. GM07552 cells contain the following known variants of CFTR: Phe508DEL, Arg553TER, and have alleles 7T / 9T. GM12785 contains known variants in the following CFTR genes: Arg347Pro, Gly551Asp, and has alleles 7T / 7T. The extracted genomic DNA (NA07552 or NA012785) was subjected to targeted amplification using 15 pairs of target-specific primers to amplify 15 different variants in the CFTR gene. Target-specific primers were mixed with the final concentration of 25 nM from Amplification buffer (magnesium-reduced version) and amplification enzyme in the Gold Single CellDNA-Seq Kit are mixed. Targeted amplification PCR was performed as follows:
[0134] 95°C for 3 minutes - 1 cycle
[0135] 95...
example 3
[0147] Example 3: Detection of CFTR mutations in clinical samples
[0148] This study was done using trophectoderm biopsy samples collected from embryos that had previously undergone traditional SNP and CNV analysis using a two-step approach in which the first biopsy was used for SNP determination followed by a second biopsy for copy number determination. this is in Figure 14 A schematic overview is given in A. Four embryos were derived from mothers identified as carriers of the pathogenic CFTR variant SNP F1052V, and from fathers identified as carriers of the R117H variant. Such as Figure 14 In A, the first biopsy revealed that embryos 3 and 4 were compound heterozygotes, carrying two pathogenic variants from the mother and father. Therefore, these two embryos were not further screened for potential copy number variations (CNVs) using a second biopsy. Embryo 1 and Embryo 2 were biopsied a second time, and potential CNV aneuploidy was identified in Embryo 1 and Embryo...
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