Question

1) You are a molecular biologist who has been asked to sequence a cancer patient’s nuclear exome. You have access to all sequencing-bysynthesis technologies on the market. Explain which method you would use for this sample and provide a summary of the approach?

b) A scientific researcher has obtained 5x coverage for a whole genome using massively parallel sequencing. Giving your reasoning, explain whether they should use this sequencing data for publication in a good quality scientific journal.

Multiple detailed answers would be greatly appreciated, trying to obtain as much info as possible (detailed responses and sources)

Answer 1

1. a Next-generation sequencing (NGS) technology

NGS has been successfully utilized for developing biomarkers to assess susceptibility, diagnosis, prognosis and treatment of cancers. NGS provides a powerful tool in the field of medical genetics, allowing one to perform multi-gene analysis and to sequence entire exomes (WES), transcriptomes or genomes (WGS). Among the various types of cancer for exmple breast cancer (BC) is the most diagnosed cancer in women worldwide. NGS technologies offer a powerful tool for the discovery of novel factors involved in familial breast/ovarian cancer. BRCA1 and BRCA2 are the main predisposing genes for hereditary breast syndrome.

The great power of NGS technology is the capability to massively sequence millions of DNA reads.  The advent of NGS is the possibility to obtain not only targeted sequencing of several genes involved in BC susceptibility, but also sequencing of entire exomes, transcriptomes or genomes for the identification of novel genes oresponsible for breast cancer predisposition. The easiest approach of NGS is targeted gene sequencing. WGS is based on the sequencing of the entire genome, provides the most complete analysis for the characterization of the genomic profile and the possible biological consequences. It leads to the discovery of new molecular alterations in coding as well as non-coding regions.

BRCA1/2 Gene analysis by NGS

In BRCA1 and BRCA2 the mutations scattered across the whole length of both genes due to the large size. The Sanger sequencing is the best approach to identify point mutations, small deletions or insertions, but it is time consuming and expensive. The advent of NGS technologies made it possible to perform multi-gene massive sequencing with very sensitive detection of gene variants (analyzing both coding and non-coding regions).  The majority of clinical samples from cancer patients, such as surgical specimens are stored in formalin‐fixed, paraffin‐embedded (FFPE) tissue, which can be used for DNA extraction and NGS analysis if processed and preserved appropriately. Flow of a NGS‐based gene panel test utilizing this FFPE tissue. Preparation and preservation of FFPE tissue is the first step of the panel test, followed by DNA extraction, NGS analysis, curation of the data and report of the data with therapeutic recommendations. It is important for appropriate tissue preservation to fix for the appropriate time with formalin. Over‐fixation results in excessive cross‐linking, thus interfering with the extraction of DNA and proteins. Biopsy samples can be very useful for NGS analysis. For example in case of breast cancer genomic analysis of tissue samples from biopsies who are treated with neoadjuvant chemotherapy will be useful for NGS analysis.

b. Massively parallel sequencing has reduced the cost and increased the throughput of genomic sequencing. The clinical use of massively parallel sequencing will provide a way to identify the cause of many diseases of unknown etiology through simultaneous screening of thousands of loci for pathogenic mutations and novel infectious agents. The advantage of massively parallel sequencing is the ability to detect minor alleles accurately. Each DNA fragment within the sequenced library is amplified and sequenced . Higher coverage is especially important when looking for mutations or sequence variants in repetitive or massively rearranged regions. For example, a single lane on an Illumina flow cell would provide more than 68× coverage of the entire genomic segment containing the DMD gene. In this case greater depth means more sequencing, thereby reducing the advantages of using massively parallel sequencing. Massively parallel sequencing could accurately measure genetic alterations due to cancer that occur in only a small fraction of the cells tested. This is because of the independent amplification and sequencing of millions of different DNA fragments from each specimen which permits the accurate detection of rare sequences if the depth of coverage is sufficient.