A guided-inquiry investigation of genetic variants using Oxford nanopore sequencing for an undergraduate molecular biology laboratory course

Abstract

Next Generation Sequencing (NGS) has become an important tool in the biological sciences and has a growing number of applications across medical fields. Currently, few undergraduate programs provide training in the design and implementation of NGS applications. Here, we describe an inquiry-based laboratory exercise for a college-level molecular biology laboratory course that uses real-time MinION deep sequencing and bioinformatics to investigate characteristic genetic variants found in cancer cell-lines. The overall goal for students was to identify non-small cell lung cancer (NSCLC) cell-lines based on their unique genomic profiles. The units described in this laboratory highlight core principles in multiplex PCR primer design, real-time deep sequencing, and bioinformatics analysis for genetic variants. We found that the MinION device is an appropriate, feasible tool that provides a comprehensive, hands-on NGS experience for undergraduates. Student evaluations demonstrated increased confidence in using molecular techniques and enhanced understanding of NGS concepts. Overall, this exercise provides a pedagogical tool for incorporating NGS approaches in the teaching laboratory as way of enhancing students' comprehension of genomic sequence analysis. Further, this NGS lab module can easily be added to a variety of lab-based courses to help undergraduate students learn current DNA sequencing methods with limited effort and cost.

Keywords: cancer; next-generation sequencing; teaching laboratory; variants.

FIGURE 1

An overview of the 4‐week module implemented at the University of Vermont to teach variant analysis with the MinION sequencing platform. (a) Students used primers they created to amplify DNA regions known to flank genetic variants in the TP53, EGFR, and KRAS gene in non‐small cell lung cancer (NSCLC) cells using both single target and multiplex PCR. A portion of the PCR‐product was then run on an agarose gel for size validation while the rest of the sample was pooled and sequenced on the MinION platform. after sequencing, students were asked to identify the starting NSCLC cell‐line by genotype analysis using integrative genome viewer (IGV). (b) Flowchart summarizing the learning objections and outcomes for the four units taught [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Class amplicon pool was sequenced on the MinION and the follow‐up bioinformatic analysis performed. (a) Genotyping assay was performed to amplify regions encompassing genomic variants identified in the TP53, EGFR, and KRAS gene (lane 3–6). A multiplex PCR reaction containing all primers of interest are shown (lane 7). A reaction which lacked genomic DNA was included as a negative control (lane 1) while primers amplifying a region in GAPDH was included as the positive control (lane 2). (b) Each forward and reverse primer used in the PCR reactions contained a 12‐nucleotide index at the 5′ end. (c) The multiplex PCR reaction carried out by each student group were then pooled into a single tube (class amplicon pool) and sequenced on the MinION. (d) Data output from the MinION sequencing run was analyzed using the bioinformatic pipeline shown [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

Quality of MinION sequencing reads. (a) The total demultiplexed reads for each amplicon sequenced is shown. (b) Plotting the average read quality versus read lengths (kbp). Each black dot represents a sequenced read that passed quality control cutoffs and were then used for downstream analysis [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

The integrated genomics viewer (IGV) was used by students to assess genomic variants in lung cancer cells. (a) Displaying the alignment of the genomic region amplified in the EGFR gene in HCC827 cells. A snapshot centered on the exon 19 deletion (15 bp) is shown. The gray horizontal bar represents a mapped read, while the lack of the gray bar indicates no reads mapped for that region. (b) Displaying the alignment of the genomic region amplified in the TP53 gene in H1975 cells. A snapshot centered on the single nucleotide polymorphism R273H is shown. Bases that map to the reference genome are shown in gray, while a base mismatch is colored in red [Color figure can be viewed at wileyonlinelibrary.com]