Assay Overview
The VeriSeq NIPT Solution v2 – RUO is an automated solution that consists of automated sample preparation and sequencing data analysis. The TG VeriSeq NIPT Sample Prep Kits are specialized single-use reagents. The kits are used with the VeriSeq NIPT Microlab STAR to prepare batches of 24, 48, or 96 libraries for next-generation sequencing (NGS). Specialized software, the Assay Control Software, analyzes whole genome, paired-end sequencing data. In addition to analyzing sequencing data, the Assay Software produces sample results, process reports, and notifications. The workflow consists of the following procedures:
| • | Sample Collection—Collect 7–10 ml of maternal peripheral whole blood in a Streck cell-free DNA Blood Collection Tube (BCT). This method of sample collection prevents cell lysis and genomic contamination by stabilizing whole blood. |
| • | Plasma Isolation—Within 5 days of blood collection, isolate plasma from maternal peripheral whole blood using standard centrifugation techniques. The ML STAR aspirates and dispenses plasma into a 96-well deep-well plate for subsequent processing. In case retesting is required, recap postprocessing samples and store at 4°C for an additional 5 days (up to a total of 10 days after blood collection). |
Exceeding the specified storage times can negatively impact individual sample failure rates.
| • | cfDNA Extraction—cfDNA from plasma is purified by absorption onto a binding plate using a vacuum manifold. The binding plate is washed and eluted to remove contaminants. |
| • | Library Preparation—Purified cfDNA fragments undergo an end repair process. Indexed adapters are then ligated onto the processed cfDNA fragments. Solid phase reverse immobilization beads purify the ligated DNA. Each sample in a set of 24, 48, or 96 receives a unique indexed adapter. The adapters serve the following purposes: |
| – | Indexes allow for sample identification in subsequent sequencing. |
| – | Index adapters contain sequences that enable the library to bind onto the solid surface of a sequencing flow cell. The flow cell is then used for cluster generation and subsequent sequencing. |
| • | Quantification—A fluorescent dye enables quantification of the library product with concentration determined by comparison to a DNA standard curve. |
| • | Library Pooling—The libraries are pooled together into one pool of 24 or 48 libraries, in adjusted amounts to minimize diversity in coverage variation. |
| • | Sequencing—Each pool is then sequenced using a NextSeq 500/550 instrument. Libraries are sequenced using 2 x 36 paired-end reads. |
| • | Analysis—Nucleotide base calls are made directly from signal intensity measurements during sequencing. Secondary analysis consists of the following steps: |
| – | Identifying library fragments by index sequence and alignment of the paired-end reads to a human reference genome. |
| – | Estimating the fetal fraction of the library by combining information from the distribution of both the lengths and genomic coordinates of the library fragments. |
| – | After accounting for known biases, a statistical model identifies regions of the genome that are under- or overrepresented in the library. The model looks for under- or overrepresentation consistent with an anomaly at the estimated level of fetal fraction. |
| – | The VeriSeq NIPT RUO Report provides summary results for the selected menu. |
| – | The Supplementary Report provides data for additional metrics based on a batch, sample, or region. Use of the Supplementary Report is discretionary and not required. |
Refer to VeriSeq NIPT Workflow Manager for information on the VeriSeq NIPT Assay Software v2 – RUO and VeriSeq NIPT Workflow Manager.
