TP53 gene mutation analysis in chronic lymphocytic leukemia by nanopore MinION sequencing

Abstract

Background: The assessment of TP53 mutational status is becoming a routine clinical practice for chronic lymphocytic leukemia patients (CLL). A broad spectrum of molecular techniques has been employed so far, including both direct Sanger sequencing and next generation sequencing. Oxford Nanopore Technologies recently released the MinION an USB-interfaced sequencer. In this paper we report our experience, with the MinION technology for the detection of the TP53 gene mutation in CLL patients. Twelve CLL patients at diagnosis were included in this study. All except one patient showed the TP53 gene deletion in Fluorescence in situ hybridization experiments. Patients were investigated for TP53 mutation by Sanger and by MinION sequencing. Analysis by Sanger was performed according with the IARC protocol. Analysis by MinION was performed adopting a strategy based on long template PCR, read error correction, and post variant calling filtering.

Results: Due to the high error rate of nanopore technology, sequence data were both used directly and before correction with two different in silico methods: ALEC and nanocorrect. A mean error rate of 15 % was detected before correction that was reduced to 4-5 % after correction. Analysis by Sanger sequencing was able to detect four patients mutated for TP53. MinION analysis detected one more mutated patient previously not detected from Sanger.

Conclusion: In our hands, the Nanopore technology shows correlation with Sanger sequencing but more sensitive, manageable and less expensive, and therefore has proven to be a useful tool for TP53 gene mutation detection.

Keywords: Chronic Lymphocytic Leukemia; MinION; Nanopore; Sequencing; TP53.

Fig. 1

Reads length distribution plot

Fig. 2

Coverage plots from corrected and uncorrected (Raw) reads after mapping on TP53 genomic sequence

Fig. 3

Example of mutations detected in two samples (CLL#8 and CLL#12). The mutation detected in CLL#8 was not found from nanocorrect corrected reads. CLL#12 mutation was detected only from the analysis with raw and ALEC corrected reads. The same mutation was barely visible in Sanger sequencing. Aligned reads are visualized by IGV

Fig. 4

Schematic workflow implemented for TP53 mutational analysis