Next generation sequencing

INTRODUCTION
 Next-generation sequencing (NGS) technology has expanded in the
last decades with enhancement in the reliability, sequencing
chemistry, pipeline analyses, data interpretation and costs.
 Such advances make the use of NGS feasible in clinical practice .
This review describes the recent technological developments in
NGS applied to the field of oncology.
 Next-generation sequencing (NGS) is also called massive parallel
sequencing
 Some advantages of NGS sequencers are the high-throughput
sequencing capacity of large genomic regions or small regions for
many samples.
 Nowadays, the use of NGS almost replaced conventional Sanger
sequencing and is a very versatile approach for several clinical and
non-clinical applications.
 It is very cost effective

AIM
 The present review describes the major factor in NGS technology, the technical
developments and application of NGS to the field of oncology, i.e., hereditary
cancer syndromes and sporadic cancer and it’s diagnostics
 NGS studies exploring the somatic mutation profile in cancer .
 In clinical diagnostics, in order to identify familial germline mutations, WGS may
be useful in case genetic tests based on WES returned a negative result in families
with a high probability of carrying a genetic mutation.
 A thorough overview of the different NGS platforms and approaches e.g. Illumina
 Current NGS technology is sorted in two major types, i.e., short- and long-read
sequencing
 Short-read sequencing has low costs per Gb and high accuracy (low final error-rate;
0.1 Kb) and has been the method most frequently used .
 In contrast, the low accuracy (high final error-rate; >1 Kb) and the high costs per
Gb of long-read sequencing make the use of these approaches non-versatile for all-
purposes

PROCEDURE
 The initial input material can be genomic DNA (DNA-seq),
messenger or non-coding RNA (RNA-seq) or any
nucleic/ribonucleic material obtained after specific procedures.
 The implementation of NGS technology can be visualised as four
major blocks:-
1) Library preparation
2) Sequencing
3) Quality analysis
4) Data Analysis interpretation

DATA ANYLYSIS
 After raw data are assessed for sufficient quality,
data analyses and interpretation continues using
different pipelines depending on the approach used
(gene-panel, WES, WGS or targeted-RNA-seq)
 It was indicated that genes
besides BRCA1 and BRCA2 are mutated and confer
a moderate to high cancer risk
 Aloraifi and co-workers [1] sequenced 312 genes
among 104 BRCAx subjects and found that 13
subjects carried a pathogenic variant, most
frequently occurring
in ATM, RAD50, PALB2, CHEK2 and TP53

CONCLUSION
 In conclusions, liquid biopsy analyses by NGS have been applied to
several cancer types like hepatocellular carcinoma [278],
gynaecological cancer [2], lung cancer [3,5], urinary tract cancer
[271], paediatric oncology [6], gastrointestinal tumours [7] and
other types, even through WES/WGS analyses [8,9]
 The NGS approach for PGx can involve the analysis of the entire
exome (WES) or the entire genome (WGS). WGS has the main
advantage of encompassing also the non-coding regions, e.g.,
involved in regulation of transcription or affecting splicing, which is
not present in WES data
 NGS has brought unprecedented advances in understanding the
biology of diseases, with important clinical implications .

REFERENCE
1. Aloraifi F., McDevitt T., Martiniano R., McGreevy J., McLaughlin R., Egan C.M.,
Cody N., Meany M., Kenny E., Green A.J., et al. Detection of novel germline
mutations for breast cancer in non-BRCA1/2families. FEBS J. 2015;282:3424–3437.
doi: 10.1111/febs.13352. [PubMed] [Cross Ref]
2. Forshew T., Murtaza M., Parkinson C., Gale D., Tsui D.W., Kaper F., Dawson S.J.,
Piskorz A.M., Jimenez-Linan M., Bentley D., et al. Noninvasive identification and
monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci.
Transl. Med. 2012;4:136ra68. doi: 10.1126/scitranslmed.3003726. [PubMed] [Cross
Ref]
3. Couraud S., Vaca-Paniagua F., Villar S., Oliver J., Schuster T., Blanche H., Girard N.,
Tredaniel J., Guilleminault L., Gervais R., et al. Noninvasive diagnosis of actionable
mutations by deep sequencing of circulating free DNA in lung cancer from never-
smokers: A proof-of-concept study from BioCAST/IFCT-1002. Clin. Cancer
Res. 2014;20:4613–4624. doi: 10.1158/1078-0432.CCR-13-3063. [PubMed][Cross
Ref]
4. Ward D.G., Baxter L., Gordon N.S., Ott S., Savage R.S., Beggs A.D., James J.D.,
Lickiss J., Green S., Wallis Y., et al. Multiplex PCR and Next Generation Sequencing
for the Non-Invasive Detection of Bladder Cancer. PLoS ONE. 2016;11:e0149756
doi: 10.1371/journal.pone.0149756. [PMC free article][PubMed] [Cross Ref]

5. Xu S., Lou F., Wu Y., Sun D.Q., Zhang J.B., Chen W., Ye H., Liu J.H., Wei S., Zhao M.Y., et
al. Circulating tumor DNA identified by targeted sequencing in advanced-stage non-small cell
lung cancer patients. Cancer Lett. 2016;370:324–331. doi:
10.1016/j.canlet.2015.11.005. [PubMed] [Cross Ref]
6. Kurihara S., Ueda Y., Onitake Y., Sueda T., Ohta E., Morihara N., Hirano S., Irisuna F.,
Hiyama E. Circulating free DNA as non-invasive diagnostic biomarker for childhood solid
tumors. J. Pediatr. Surg. 2015;50:2094–2097. doi:
10.1016/j.jpedsurg.2015.08.033. [PubMed] [Cross Ref]
7. Ueda M., Iguchi T., Masuda T., Nakahara Y., Hirata H., Uchi R., Niida A., Momose K.,
Sakimura S., Chiba K., et al. Somatic mutations in plasma cell-free DNA are diagnostic
markers for esophageal squamous cell carcinoma recurrence. Oncotarget. 2016;7:62280–
62291. doi: 10.18632/oncotarget.11409.[PMC free article] [PubMed] [Cross Ref]
8. Gasch C., Pantel K., Riethdorf S. Whole Genome Amplification in Genomic Analysis of
Single Circulating Tumor Cells. Methods Mol. Biol. 2015;1347:221–232. [PubMed]
9. Huang L., Ma F., Chapman A., Lu S., Xie X.S. Single-Cell Whole-Genome Amplification
and Sequencing: Methodology and Applications. Annu. Rev. Genom. Hum. Genet. 2015;16:79–
102. doi: 10.1146/annurev-genom-090413-025352. [PubMed] [Cross Ref]

Next generation sequencing

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