title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, P.zza G. Cesare, 11 70124, Bari, Italy.
authors.forename: Crescenzio Francesco
authors.initials: CF
authors.lastname: Minervini
authors.affiliation: Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, P.zza G. Cesare, 11 70124, Bari, Italy.
authors.forename: Cosimo
authors.initials: C
authors.lastname: Cumbo
authors.affiliation: Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, P.zza G. Cesare, 11 70124, Bari, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Orsini
authors.affiliation: Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, P.zza G. Cesare, 11 70124, Bari, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Brunetti
authors.affiliation: Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, P.zza G. Cesare, 11 70124, Bari, Italy.
authors.forename: Luisa
authors.initials: L
authors.lastname: Anelli
authors.affiliation: Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, P.zza G. Cesare, 11 70124, Bari, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Zagaria
authors.affiliation: Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, P.zza G. Cesare, 11 70124, Bari, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Minervini
authors.affiliation: Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, P.zza G. Cesare, 11 70124, Bari, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Casieri
authors.affiliation: Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, P.zza G. Cesare, 11 70124, Bari, Italy.
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Coccaro
authors.affiliation: Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, P.zza G. Cesare, 11 70124, Bari, Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Tota
authors.affiliation: Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, P.zza G. Cesare, 11 70124, Bari, Italy.
authors.forename: Luciana
authors.initials: L
authors.lastname: Impera
authors.affiliation: Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, P.zza G. Cesare, 11 70124, Bari, Italy.
authors.forename: Annamaria
authors.initials: A
authors.lastname: Giordano
authors.affiliation: Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, P.zza G. Cesare, 11 70124, Bari, Italy.
authors.forename: Giorgina
authors.initials: G
authors.lastname: Specchia
authors.affiliation: Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, P.zza G. Cesare, 11 70124, Bari, Italy. francesco.albano@uniba.it.
authors.forename: Francesco
authors.identifier: http://orcid.org/0000-0001-7926-6052
authors.initials: F
authors.lastname: Albano
mesh_terms: D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000235:genetics*; D003362:Cost-Benefit Analysis; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods*; D005260:Female; D017048:Health Care Costs; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000191:economics / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53
keywords: Chronic Lymphocytic Leukemia; MinION; Nanopore; Sequencing; TP53