Assessing copy number alterations in targeted, amplicon-based next-generation sequencing data

Catherine Grasso, Timothy Butler, Katherine Rhodes, Michael Quist, Tanaya L. Neff, Stephen Moore, Scott A. Tomlins, Erica Reinig, Carol Beadling, Mark Andersen, Christopher Corless

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

Changes in gene copy number are important in the setting of precision medicine. Recent studies have established that copy number alterations (CNAs) can be detected in sequencing libraries prepared by hybridization-capture, but there has been comparatively little attention given to CNA assessment in amplicon-based libraries prepared by PCR. In this study, we developed an algorithm for detecting CNAs in amplicon-based sequencing data. CNAs determined from the algorithm mirrored those from a hybridization-capture library. In addition, analysis of 14 pairs of matched normal and breast carcinoma tissues revealed that sequence data pooled from normal samples could be substituted for a matched normal tissue without affecting the detection of clinically relevant CNAs (>|2| copies). Comparison of CNAs identified by array comparative genomic hybridization and amplicon-based libraries across 10 breast carcinoma samples showed an excellent correlation. The CNA algorithm also compared favorably with fluorescence in situ hybridization, with agreement in 33 of 38 assessments across four different genes. Factors that influenced the detection of CNAs included the number of amplicons per gene, the average read depth, and, most important, the proportion of tumor within the sample. Our results show that CNAs can be identified in amplicon-based targeted sequencing data, and that their detection can be optimized by ensuring adequate tumor content and read coverage.

Original languageEnglish (US)
Pages (from-to)53-63
Number of pages11
JournalJournal of Molecular Diagnostics
Volume17
Issue number1
DOIs
StatePublished - Jan 1 2015

Fingerprint

Libraries
Breast Neoplasms
Matched-Pair Analysis
Precision Medicine
Comparative Genomic Hybridization
Gene Dosage
Fluorescence In Situ Hybridization
Genes
Neoplasms
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Molecular Medicine
  • Pathology and Forensic Medicine

Cite this

Assessing copy number alterations in targeted, amplicon-based next-generation sequencing data. / Grasso, Catherine; Butler, Timothy; Rhodes, Katherine; Quist, Michael; Neff, Tanaya L.; Moore, Stephen; Tomlins, Scott A.; Reinig, Erica; Beadling, Carol; Andersen, Mark; Corless, Christopher.

In: Journal of Molecular Diagnostics, Vol. 17, No. 1, 01.01.2015, p. 53-63.

Research output: Contribution to journalArticle

Grasso, Catherine ; Butler, Timothy ; Rhodes, Katherine ; Quist, Michael ; Neff, Tanaya L. ; Moore, Stephen ; Tomlins, Scott A. ; Reinig, Erica ; Beadling, Carol ; Andersen, Mark ; Corless, Christopher. / Assessing copy number alterations in targeted, amplicon-based next-generation sequencing data. In: Journal of Molecular Diagnostics. 2015 ; Vol. 17, No. 1. pp. 53-63.
@article{b81ae4c73088485dbcda677e5180d04e,
title = "Assessing copy number alterations in targeted, amplicon-based next-generation sequencing data",
abstract = "Changes in gene copy number are important in the setting of precision medicine. Recent studies have established that copy number alterations (CNAs) can be detected in sequencing libraries prepared by hybridization-capture, but there has been comparatively little attention given to CNA assessment in amplicon-based libraries prepared by PCR. In this study, we developed an algorithm for detecting CNAs in amplicon-based sequencing data. CNAs determined from the algorithm mirrored those from a hybridization-capture library. In addition, analysis of 14 pairs of matched normal and breast carcinoma tissues revealed that sequence data pooled from normal samples could be substituted for a matched normal tissue without affecting the detection of clinically relevant CNAs (>|2| copies). Comparison of CNAs identified by array comparative genomic hybridization and amplicon-based libraries across 10 breast carcinoma samples showed an excellent correlation. The CNA algorithm also compared favorably with fluorescence in situ hybridization, with agreement in 33 of 38 assessments across four different genes. Factors that influenced the detection of CNAs included the number of amplicons per gene, the average read depth, and, most important, the proportion of tumor within the sample. Our results show that CNAs can be identified in amplicon-based targeted sequencing data, and that their detection can be optimized by ensuring adequate tumor content and read coverage.",
author = "Catherine Grasso and Timothy Butler and Katherine Rhodes and Michael Quist and Neff, {Tanaya L.} and Stephen Moore and Tomlins, {Scott A.} and Erica Reinig and Carol Beadling and Mark Andersen and Christopher Corless",
year = "2015",
month = "1",
day = "1",
doi = "10.1016/j.jmoldx.2014.09.008",
language = "English (US)",
volume = "17",
pages = "53--63",
journal = "Journal of Molecular Diagnostics",
issn = "1525-1578",
publisher = "Association of Molecular Pathology",
number = "1",

}

TY - JOUR

T1 - Assessing copy number alterations in targeted, amplicon-based next-generation sequencing data

AU - Grasso, Catherine

AU - Butler, Timothy

AU - Rhodes, Katherine

AU - Quist, Michael

AU - Neff, Tanaya L.

AU - Moore, Stephen

AU - Tomlins, Scott A.

AU - Reinig, Erica

AU - Beadling, Carol

AU - Andersen, Mark

AU - Corless, Christopher

PY - 2015/1/1

Y1 - 2015/1/1

N2 - Changes in gene copy number are important in the setting of precision medicine. Recent studies have established that copy number alterations (CNAs) can be detected in sequencing libraries prepared by hybridization-capture, but there has been comparatively little attention given to CNA assessment in amplicon-based libraries prepared by PCR. In this study, we developed an algorithm for detecting CNAs in amplicon-based sequencing data. CNAs determined from the algorithm mirrored those from a hybridization-capture library. In addition, analysis of 14 pairs of matched normal and breast carcinoma tissues revealed that sequence data pooled from normal samples could be substituted for a matched normal tissue without affecting the detection of clinically relevant CNAs (>|2| copies). Comparison of CNAs identified by array comparative genomic hybridization and amplicon-based libraries across 10 breast carcinoma samples showed an excellent correlation. The CNA algorithm also compared favorably with fluorescence in situ hybridization, with agreement in 33 of 38 assessments across four different genes. Factors that influenced the detection of CNAs included the number of amplicons per gene, the average read depth, and, most important, the proportion of tumor within the sample. Our results show that CNAs can be identified in amplicon-based targeted sequencing data, and that their detection can be optimized by ensuring adequate tumor content and read coverage.

AB - Changes in gene copy number are important in the setting of precision medicine. Recent studies have established that copy number alterations (CNAs) can be detected in sequencing libraries prepared by hybridization-capture, but there has been comparatively little attention given to CNA assessment in amplicon-based libraries prepared by PCR. In this study, we developed an algorithm for detecting CNAs in amplicon-based sequencing data. CNAs determined from the algorithm mirrored those from a hybridization-capture library. In addition, analysis of 14 pairs of matched normal and breast carcinoma tissues revealed that sequence data pooled from normal samples could be substituted for a matched normal tissue without affecting the detection of clinically relevant CNAs (>|2| copies). Comparison of CNAs identified by array comparative genomic hybridization and amplicon-based libraries across 10 breast carcinoma samples showed an excellent correlation. The CNA algorithm also compared favorably with fluorescence in situ hybridization, with agreement in 33 of 38 assessments across four different genes. Factors that influenced the detection of CNAs included the number of amplicons per gene, the average read depth, and, most important, the proportion of tumor within the sample. Our results show that CNAs can be identified in amplicon-based targeted sequencing data, and that their detection can be optimized by ensuring adequate tumor content and read coverage.

UR - http://www.scopus.com/inward/record.url?scp=84964310688&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84964310688&partnerID=8YFLogxK

U2 - 10.1016/j.jmoldx.2014.09.008

DO - 10.1016/j.jmoldx.2014.09.008

M3 - Article

C2 - 25468433

AN - SCOPUS:84964310688

VL - 17

SP - 53

EP - 63

JO - Journal of Molecular Diagnostics

JF - Journal of Molecular Diagnostics

SN - 1525-1578

IS - 1

ER -