High-throughput sequencing: a failure mode analysis

George S Yang,Sarah A Barber,Miruna Balasundaram,Duane Smailus,Marco A Marra,Robert A Holt,Jeffery M Stott

doi:10.1186/1471-2164-6-2

George S Yang, Sarah A Barber + Show 5 more

Open Access

https://doi.org/10.1186/1471-2164-6-2

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Journal: BMC Genomics	Publication Date: Jan 4, 2005
Citations: 19	License type: CC BY 2.0

Affiliation: Canada's Michael Smith Genome Sciences Centre

Abstract

BackgroundBasic manufacturing principles are becoming increasingly important in high-throughput sequencing facilities where there is a constant drive to increase quality, increase efficiency, and decrease operating costs. While high-throughput centres report failure rates typically on the order of 10%, the causes of sporadic sequencing failures are seldom analyzed in detail and have not, in the past, been formally reported.ResultsHere we report the results of a failure mode analysis of our production sequencing facility based on detailed evaluation of 9,216 ESTs generated from two cDNA libraries. Two categories of failures are described; process-related failures (failures due to equipment or sample handling) and template-related failures (failures that are revealed by close inspection of electropherograms and are likely due to properties of the template DNA sequence itself).ConclusionsPreventative action based on a detailed understanding of failure modes is likely to improve the performance of other production sequencing pipelines.

Highlights

Basic manufacturing principles are becoming increasingly important in highthroughput sequencing facilities where there is a constant drive to increase quality, increase efficiency, and decrease operating costs
Preventative action based on a detailed understanding of failure modes is likely to improve the performance of other production sequencing pipelines
A Failure Mode Analysis (FMA) strategy was developed to determine the likely causes of sporadic unsuccessful sequence reads

Summary

Introduction

Basic manufacturing principles are becoming increasingly important in highthroughput sequencing facilities where there is a constant drive to increase quality, increase efficiency, and decrease operating costs. While high-throughput centres report failure rates typically on the order of 10%, the causes of sporadic sequencing failures are seldom analyzed in detail and have not, in the past, been formally reported. Highthroughput sequencing facilities are focused on establishing automated procedures that maintain long read length and high overall success rates. Sequencing centres, monitor sequencing success on a larger scale referencing overall pass rates and average read lengths, typically in terms of Phred 20 bases [2]. A Failure Mode Analysis (FMA) strategy was developed to determine the likely causes of sporadic unsuccessful sequence reads. We systematically examine these failed reads in the context of a high-throughput sequencing pipeline to establish the mode and frequency of each type of failure. As of December 8, 2004, we have generated 1,263,904,347 Q20 bases using our 384-well culturing, DNA preparation, and cycle (page number not for citation purposes)

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