Abstract
Next Generation Sequencing (NGS) is a powerful tool getting into the field of clinical examination. Its preliminary application in pre-implantation comprehensive chromosomal screening (PCCS) of assisted reproduction (test-tube baby) has shown encouraging outcomes that improves the success rate of in vitro fertilization. However, the conventional NGS library construction is time consuming. In addition with the whole genome amplification (WGA) procedure in prior, makes the single cell NGS assay hardly be accomplished within an adequately short turnover time in supporting fresh embryo implantation. In this work, we established a concise single cell sequencing protocol, ChromInst, in which the single cell WGA and NGS library construction were integrated into a two-step PCR procedure of ~ 2.5hours reaction time. We then validated the feasibility of ChromInst for overnight PCCS assay by examining 14 voluntary donated embryo biopsy samples in a single sequencing run of Miseq with merely 13M reads production. The good compatibility of ChromInst with the restriction of Illumina sequencing technique along with the good library yield uniformity resulted superior data usage efficiency and reads distribution evenness that ensures precisely distinguish of 6 normal embryos from 8 abnormal one with variable chromosomal aneuploidy. The superior succinctness and effectiveness of this protocol permits its utilization in other time limited single cell NGS applications.
Highlights
With wide application of PCR, FISH and DNA chip technology, medical examination has moved into the age of molecular diagnosis in the past decade
Fifteen single cells were used in the test
To minimize examination turnover time, we pooled the library from each sample at equal volume instead of the conventional pooling strategy that performs qPCR quantification to each library pools them in equal molar manner
Summary
The strategy of single cell WGA in this work was adapted from Multiple Annealing and Looping Based Amplification Cycles (MALBAC) method [34], while the chemical components of cell lysis, pre-amplification, exponential amplification and thermal cycling program remain the same. The pre-amplification primers were modified (Table 1) to be compatible with the adaptor sequences of Illumina sequencing library (Illumina, San Diego, USA, Table 1). A 30 μl of preamplification mixture containing pre-amplification primers and DNA Polymerase was added to the reaction. After the 12 cycles of pre-amplification program (95 ̊C-2min, 95 ̊C-15s, 15 ̊C50s, 25 ̊C-40s, 35 ̊C-30s, 65 ̊C-40s and 75 ̊C-40s), another 30 μl of exponential amplification mix containing exponential amplification primers was added and subjected for 17 cycles of exponential amplification (94 ̊C-30s, 94 ̊C-20s, 63 ̊C-30s and 72 ̊C-40s). The resulting WGA product with the format of Illumina NGS sequencing library (Fig 1) was subject to be sequenced on Illumina platform
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