Targeted Sequencing Approach and Its Clinical Applications for the Molecular Diagnosis of Human Diseases

Xiao Meng Pei, Martin Ho Yin Yeung, Alex Ngai Nick Wong, Hin Fung Tsang, Allen Chi Shing Yu, Aldrin Kay Yuen Yim, Sze Chuen Cesar Wong, Xiao Meng Pei, Martin Ho Yin Yeung, Alex Ngai Nick Wong, Hin Fung Tsang, Allen Chi Shing Yu, Aldrin Kay Yuen Yim, Sze Chuen Cesar Wong

Abstract

The outbreak of COVID-19 has positively impacted the NGS market recently. Targeted sequencing (TS) has become an important routine technique in both clinical and research settings, with advantages including high confidence and accuracy, a reasonable turnaround time, relatively low cost, and fewer data burdens with the level of bioinformatics or computational demand. Since there are no clear consensus guidelines on the wide range of next-generation sequencing (NGS) platforms and techniques, there is a vital need for researchers and clinicians to develop efficient approaches, especially for the molecular diagnosis of diseases in the emergency of the disease and the global pandemic outbreak of COVID-19. In this review, we aim to summarize different methods of TS, demonstrate parameters for TS assay designs, illustrate different TS panels, discuss their limitations, and present the challenges of TS concerning their clinical application for the molecular diagnosis of human diseases.

Keywords: COVID-19 detection; bacteria identification; cancer marker detection; molecular diagnosis; next-generation sequencing; targeted sequencing.

Conflict of interest statement

A.C.S.Y. and A.K.Y.Y. are employed by Codex Genetics Limited. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. No conflicts of interest were declared by all the other authors.

Figures

Figure 1
Figure 1
General workflow of a NGS experiment [6]. Firstly, nucleic acid extraction and isolation is performed on specimen types, including blood and FFPE. Next, the library preparation is performed, and it includes fragmenting, performing end repairs, and the addition of adaptors to the nucleic acid fragments. Target enrichment is highly important when performing targeted sequencing to enrich the genes of interest. For sequencing, the right choice of platform, such as the number of samples per sequencing run, desired read length, and level of coverage for the assay, and using paired-end or single reads also are critical factors to affect the level of coverage, and the cost of the assay is taken into consideration prior sequencing. Lastly, the methods used in the bioinformatics analysis in the designed pipeline, including alignment, variant calling, and tertiary analyses, will be conducted for interpretation and application.
Figure 2
Figure 2
Illustration of (A–C) index hopping and (D) the resultant misalignment for two different samples (libraries). (A) During the library preparation, the unique Illumina i5 and i7 indexes would prepare and attach to individual sample DNA fragments. (B) After each sample had been uniquely indexed, the two samples can be mixed and ready for sequencing. (C) During sequencing, the demultiplexing algorithm would read the sample i5/i7 index and the indexes. Once all the indexes were finished reading, the sample read would be available for the downstream data analysis. (D) Through index hopping processing, some i5 or i7 indexes could be wound up across samples and affect the reading. Such a misalignment of sample reading would interfere with the actual interpretation of the results in a downstream bioinformatic data analysis.
Figure 3
Figure 3
Combinatorial indexing and index hopping in a targeted sequencing panel [131]. (A) In a combinatorial index plate, i5 indexes are the same across the column, while the i7 index is the same across the row. (B) As a practice avoiding index carryover if A1 to C2 (6 wells in red rectangles) are adopted as indexes for a previous NGS run. Rows A to C, whose i5 indexes are the same (30 wells in blue), and columns 1 to 2, whose i7 indexes are the same (10 wells in blue), should be avoided. Indexes D3 to E6 (8 wells in a green rectangles) share a non-repetition of neither i5 nor i7 of the previous run and, hence, can be used for the current NGS run. (C) In the PKD1/PKD2-targeted sequencing panel, the differentiation of PKD1 and PKD2 of the same patient sample can be done by a 2-plex index, which PKD1 barcoded with well A1 and PKD2 barcoded with well A2. If index hopping occurs, the PKD1 read misligated with PKD2 index may be mapped as PKD2 is read, leading to the misalignment or false positive of a heterozygous variant.

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