Factors affecting the success of next-generation sequencing in cytology specimens

Sinchita Roy-Chowdhuri; Rashmi S Goswami; Hui Chen; Keyur P Patel; Mark J Routbort; Rajesh R Singh; Russell R Broaddus; Bedia A Barkoh; Jawad Manekia; Hui Yao; L Jeffrey Medeiros; Gregg Staerkel; Rajyalakshmi Luthra; John Stewart

doi:10.1002/cncy.21597

Factors affecting the success of next-generation sequencing in cytology specimens

Sinchita Roy-Chowdhuri, Rashmi S Goswami, Hui Chen, Keyur P Patel, Mark J Routbort, Rajesh R Singh, Russell R Broaddus, Bedia A Barkoh, Jawad Manekia, Hui Yao, L Jeffrey Medeiros, Gregg Staerkel, Rajyalakshmi Luthra, John Stewart

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Abstract

BACKGROUND: The use of cytology specimens for next-generation sequencing (NGS) is particularly challenging because of the unconventional substrate of smears and the often limited sample volume. An analysis of factors affecting NGS testing in cytologic samples may help to increase the frequency of successful testing.

METHODS: This study reviewed variables associated with all in-house cytology cases (n = 207) that were analyzed by NGS with the Ion Torrent platform during a 10-month interval. A statistical analysis was performed to measure the effects of the DNA input threshold, specimen preparation, slide type, tumor fraction, DNA yield, and cytopathologist bias.

RESULTS: One hundred sixty-four of 207 cases (79%) were successfully sequenced by NGS; 43 (21%) failed because of either a low DNA yield or a template/library preparation failure. The median estimated tumor fraction and DNA concentration for the successfully sequenced cases were 70% and 2.5 ng/μL, respectively, whereas they were 60% and 0.2 ng/μL, respectively, for NGS failures. Cell block sections were tested in 91 cases, and smears were used in 116 cases. NGS success positively correlated with the DNA yield but not the tumor fraction. Cell block preparations showed a higher success rate than smears. Frosted-tip slides yielded significantly more DNA than fully frosted slides. Lowering the input DNA concentration below the manufacturer's recommended threshold of 10 ng (>0.85 ng/μL) resulted in a marked increase in the NGS success rate from 58.6% to 89.8%.

CONCLUSIONS: The failure of NGS with cytology samples is usually a result of suboptimal DNA due to multiple pre-analytical factors. Knowledge of these factors will allow better selection of cytology material for mutational analysis. Cancer (Cancer Cytopathol) 2015. © 2015 American Cancer Society.

Original language	English (US)
Journal	Cancer cytopathology
DOIs	https://doi.org/10.1002/cncy.21597
State	Published - Jul 31 2015

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10.1002/cncy.21597

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@article{d0fdc18022d342c6aec9a20f0bc0acad,

title = "Factors affecting the success of next-generation sequencing in cytology specimens",

abstract = "BACKGROUND: The use of cytology specimens for next-generation sequencing (NGS) is particularly challenging because of the unconventional substrate of smears and the often limited sample volume. An analysis of factors affecting NGS testing in cytologic samples may help to increase the frequency of successful testing.METHODS: This study reviewed variables associated with all in-house cytology cases (n = 207) that were analyzed by NGS with the Ion Torrent platform during a 10-month interval. A statistical analysis was performed to measure the effects of the DNA input threshold, specimen preparation, slide type, tumor fraction, DNA yield, and cytopathologist bias.RESULTS: One hundred sixty-four of 207 cases (79%) were successfully sequenced by NGS; 43 (21%) failed because of either a low DNA yield or a template/library preparation failure. The median estimated tumor fraction and DNA concentration for the successfully sequenced cases were 70% and 2.5 ng/μL, respectively, whereas they were 60% and 0.2 ng/μL, respectively, for NGS failures. Cell block sections were tested in 91 cases, and smears were used in 116 cases. NGS success positively correlated with the DNA yield but not the tumor fraction. Cell block preparations showed a higher success rate than smears. Frosted-tip slides yielded significantly more DNA than fully frosted slides. Lowering the input DNA concentration below the manufacturer's recommended threshold of 10 ng (>0.85 ng/μL) resulted in a marked increase in the NGS success rate from 58.6% to 89.8%.CONCLUSIONS: The failure of NGS with cytology samples is usually a result of suboptimal DNA due to multiple pre-analytical factors. Knowledge of these factors will allow better selection of cytology material for mutational analysis. Cancer (Cancer Cytopathol) 2015. {\textcopyright} 2015 American Cancer Society.",

author = "Sinchita Roy-Chowdhuri and Goswami, {Rashmi S} and Hui Chen and Patel, {Keyur P} and Routbort, {Mark J} and Singh, {Rajesh R} and Broaddus, {Russell R} and Barkoh, {Bedia A} and Jawad Manekia and Hui Yao and Medeiros, {L Jeffrey} and Gregg Staerkel and Rajyalakshmi Luthra and John Stewart",

note = "{\textcopyright} 2015 American Cancer Society.",

year = "2015",

month = jul,

day = "31",

doi = "10.1002/cncy.21597",

language = "English (US)",

journal = "Cancer cytopathology",

issn = "1934-662X",

publisher = "Wiley-Blackwell",

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TY - JOUR

T1 - Factors affecting the success of next-generation sequencing in cytology specimens

AU - Roy-Chowdhuri, Sinchita

AU - Goswami, Rashmi S

AU - Chen, Hui

AU - Patel, Keyur P

AU - Routbort, Mark J

AU - Singh, Rajesh R

AU - Broaddus, Russell R

AU - Barkoh, Bedia A

AU - Manekia, Jawad

AU - Yao, Hui

AU - Medeiros, L Jeffrey

AU - Staerkel, Gregg

AU - Luthra, Rajyalakshmi

AU - Stewart, John

PY - 2015/7/31

Y1 - 2015/7/31

N2 - BACKGROUND: The use of cytology specimens for next-generation sequencing (NGS) is particularly challenging because of the unconventional substrate of smears and the often limited sample volume. An analysis of factors affecting NGS testing in cytologic samples may help to increase the frequency of successful testing.METHODS: This study reviewed variables associated with all in-house cytology cases (n = 207) that were analyzed by NGS with the Ion Torrent platform during a 10-month interval. A statistical analysis was performed to measure the effects of the DNA input threshold, specimen preparation, slide type, tumor fraction, DNA yield, and cytopathologist bias.RESULTS: One hundred sixty-four of 207 cases (79%) were successfully sequenced by NGS; 43 (21%) failed because of either a low DNA yield or a template/library preparation failure. The median estimated tumor fraction and DNA concentration for the successfully sequenced cases were 70% and 2.5 ng/μL, respectively, whereas they were 60% and 0.2 ng/μL, respectively, for NGS failures. Cell block sections were tested in 91 cases, and smears were used in 116 cases. NGS success positively correlated with the DNA yield but not the tumor fraction. Cell block preparations showed a higher success rate than smears. Frosted-tip slides yielded significantly more DNA than fully frosted slides. Lowering the input DNA concentration below the manufacturer's recommended threshold of 10 ng (>0.85 ng/μL) resulted in a marked increase in the NGS success rate from 58.6% to 89.8%.CONCLUSIONS: The failure of NGS with cytology samples is usually a result of suboptimal DNA due to multiple pre-analytical factors. Knowledge of these factors will allow better selection of cytology material for mutational analysis. Cancer (Cancer Cytopathol) 2015. © 2015 American Cancer Society.

AB - BACKGROUND: The use of cytology specimens for next-generation sequencing (NGS) is particularly challenging because of the unconventional substrate of smears and the often limited sample volume. An analysis of factors affecting NGS testing in cytologic samples may help to increase the frequency of successful testing.METHODS: This study reviewed variables associated with all in-house cytology cases (n = 207) that were analyzed by NGS with the Ion Torrent platform during a 10-month interval. A statistical analysis was performed to measure the effects of the DNA input threshold, specimen preparation, slide type, tumor fraction, DNA yield, and cytopathologist bias.RESULTS: One hundred sixty-four of 207 cases (79%) were successfully sequenced by NGS; 43 (21%) failed because of either a low DNA yield or a template/library preparation failure. The median estimated tumor fraction and DNA concentration for the successfully sequenced cases were 70% and 2.5 ng/μL, respectively, whereas they were 60% and 0.2 ng/μL, respectively, for NGS failures. Cell block sections were tested in 91 cases, and smears were used in 116 cases. NGS success positively correlated with the DNA yield but not the tumor fraction. Cell block preparations showed a higher success rate than smears. Frosted-tip slides yielded significantly more DNA than fully frosted slides. Lowering the input DNA concentration below the manufacturer's recommended threshold of 10 ng (>0.85 ng/μL) resulted in a marked increase in the NGS success rate from 58.6% to 89.8%.CONCLUSIONS: The failure of NGS with cytology samples is usually a result of suboptimal DNA due to multiple pre-analytical factors. Knowledge of these factors will allow better selection of cytology material for mutational analysis. Cancer (Cancer Cytopathol) 2015. © 2015 American Cancer Society.

U2 - 10.1002/cncy.21597

DO - 10.1002/cncy.21597

M3 - Article

C2 - 26230354

SN - 1934-662X

JO - Cancer cytopathology

JF - Cancer cytopathology

ER -

Factors affecting the success of next-generation sequencing in cytology specimens

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