Performance Comparison of Benchtop Next-generation Sequencing Systems

Hee Sam Na

doi:10.4167/jbv.2014.44.2.208

Journal List > J Bacteriol Virol > v.44(2) > 1034133

Go to TopGo to Top Go to BottomGo to Bottom

TOOLS

Na: Performance Comparison of Benchtop Next-generation Sequencing Systems

Letter to the Editor

J Bacteriol Virol 2014;44(2):208-213.

Published online: 9 February 2014

DOI: https://doi.org/10.4167/jbv.2014.44.2.208

Performance Comparison of Benchtop Next-generation Sequencing Systems

Hee Sam Na^*

Department of Oral Microbiology, School of Dentistry, Pusan National University, Yangsan, Korea

^*Corresponding author: Hee Sam Na. Department of Oral Microbiology, School of Dentistry, Pusan National University, Yangsan, 626-810, Korea. Phone: +82-51-510-8252, Fax: +82-51-510-8246, e-mail: heesamy@pusan.ac.kr

^** This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Ministry of Education, Science and Technology (NRF-2011-0013215). The authors have no financial conflicts of interest.

Received 21 March 20147 April 2014 Accepted 9 April 2014

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

With fast development and wide applications of next generation sequencing (NGS), genomic sequence information is within reach to various research fields. Three benchtop NGS instruments are now available. The 454 GS Junior (Roche), Ion PGM (Life Technologies) and MiSeq (Illumina) are laser-printer sized and offer modest set-up and running costs. By reviewing 2 studies that compared the performance of these instruments, the major characteristics of each benchtop platforms are compared to enable direct comparisons. The 454 GS Junior generated the longest reads and most contiguous assemblies but had the lowest throughput. The Ion Torrent PGM had the highest throughput and fastest run time. The MiSeq had the highest throughput per run and lowest error rates. The Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors. Although all the platforms allow multiplexing of samples, details of experimental design, library preparation and data analysis may constrain the options. The features of the platforms provide opportunities both to conduct groundbreaking studies and to waste money. Thus, careful considerations should be made before purchasing or using any of them.

Keywords: Next generation sequencing, 454 GS Junior, Ion torrent, MiSeq

REFERENCES

1). Liu L, Li Y, Li S, Hu N, He Y, Pong R, et al. Comparison of next-generation sequencing systems. J Biomed Biotechnol. 2012; 2012:251–364.

2). Loman NJ, Misra RV, Dallman TJ, Constantinidou C, Gharbia SE, Wain J, et al. Performance comparison of benchtop high-throughput sequencing platforms. Nat Biotechnol. 2012; 30:434–9.

3). Jünemann S, Sedlazeck FJ, Prior K, Albersmeier A, John U, Kalinowski J, et al. Updating benchtop sequencing performance comparison. Nat Biotechnol. 2013; 31:294–6.

4). Koshimizu E, Miyatake S, Okamoto N, Nakashima M, Tsurusaki Y, Miyake N, et al. Performance comparison of bench-top next generation sequencers using microdroplet PCR-based enrichment for targeted sequencing in patients with autism spectrum disorder. PLoS One. 2013; 8:e74167.

5). Metzker ML. Sequencing technologies - the next generation. Nat Rev Genet. 2010; 11:31–46.

6). Glenn TC. Field guide to next-generation DNA sequencers. Mol Ecol Resour. 2011; 11:759–69.

7). Products - Technology: 454 Life Sciences, a Roche Company. 2014. http://my454.com/products/technology.asp.

8). Flusberg BA, Webster DR, Lee JH, Travers KJ, Olivares EC, Clark TA, et al. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods. 2010; 7:461–5.

9). Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, Brown CG, et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature. 2008; 456:53–9.

10). Sanders SJ, Ercan-Sencicek AG, Hus V, Luo R, Murtha MT, Moreno-De-Luca D, et al. Multiple recurrent de novo CNVs, including duplications of the 7q11.23 Williams syndrome region, are strongly associated with autism. Neuron. 2011; 70:863–85.

11). Beecham J. Annual Research Review: Child and adolescent mental health interventions: a review of progress in economic studies across different disorders. J Child Psychol Psychiatry. 2014.

12). Klei L, Sanders SJ, Murtha MT, Hus V, Lowe JK, Willsey AJ, et al. Common genetic variants, acting additively, are a major source of risk for autism. Mol Autism. 2012; 3:9.

13). Sanders SJ, Murtha MT, Gupta AR, Murdoch JD, Raubeson MJ, Willsey AJ, et al. De novo mutations revealed by whole-exome sequencing are strongly associated with autism. Nature. 2012; 485:237–41.

Table 1.

Comparison of benchtop instruments and sequencing runs (modified from reference # 2)

Platform	List price	Cost per run	Throughput (Read length)	Run time	Cost/Mb	Mb/h
454 GS Junior	$108,000	$1,000	35 Mb (400 bp)	8 hr	$31	4.4
Ion PGM
(314 chip)	$80,490	$225	10 Mb (100 bp)	3 hr	$22.5	3.3
(316 chip)		$425	100 Mb (100 bp)	3 hr	$4.25	33
(318 chip)		$625	1,000 Mb (100 bp)	3 hr	$0.63	333
MiSeq	$125,000	$750	1,500 Mb (2 × 150 bp)	27 hr	$0.5	55.5

Table 2.

Run and alignment metrics for benchtop sequencers (modified from reference # 2)

Platform	Number of reads	Total bases	Modal read length in bases	Mean read length in bases (s.d.)	Alignment coverage
Platform	Number of reads	Total bases	Modal read length in bases	Mean read length in bases (s.d.)	Chromosome	Large plasmid	Reads aligned (%)
454 GS Junior	135,992	70,999,968	518	522 (46)	11.50	5.66	99
Ion PGM (316 chip)	2,154,577	260,017,346	123	121 (16)	39.33	43.80	89
MiSeq	11,708,156	1,652,529,000	150	141 (22)	−	−	−
MiSeq demultiplexed strain 280	1,766,516	250,356,566	150	141 (21)	22.11	625.46	99

Metrics for each sequencing run are shown as well as results of alignment against the reference sequence. Depth of coverage for the chromosome and two large plasmids (pESBL and pAA) are shown with the percentage of read that align. For the MiSeq run, the sequence metrics are shown for the entire run as well as the results of de-multiplexing E. coli O104:H4 strain 280.

Table 3.

Insertion/deletion and substitution errors on read level for benchtop sequencer (modified from reference # 3)

Plateform	Sequencing kit	Indels per 100 bp	Indels per read	Substitution per 100 bp	Substitution per read
454 GS Junior	GSJ titanium	0.4011	1.8351	0.0543	0.2484
Ion PGM	100 bp	0.3520	0.3878	0.0929	0.1024
	200 bp	0.3955	0.6811	0.0303	0.0521
	300 bp	0.7054	1.4457	0.0861	0.1765
MiSeq	Nextera	0.0009	0.0013	0.0921	0.1318

Table 4.

Comparison between Ion PGM and MiSeq sequencing performance in 10 positive controls (modified from reference # 4)

	Ion PGM (TMAP^*)	MiSeq
Total read (Mb)	295.97	469.42
Average read length (bp)	116	150
% mapped on human genome	96.8%	75%
% on target regions	26.7%	22.7%
Mean depth of coverage	63	95
% of target regions at > 10-fold coverage	93.7%	96.8%
% of target regions at > 20-fold coverage	85.9%	93.2%

^* TMAP (Torrent Mapping Alignment Program) is a customized mapping tools for sequencing data generated by PGM, ignoring the indel calls around homopolymer stretch to reduce the hundreds of false negative calls.

Table 5.

Validation for mutation detection (modified from reference # 4)

	Ion PGM (TMAP)	MiSeq
Detection (+/total)	7/10	10/10
Coverage (range)	36.8 ± 21.8 (8∼77)	92.4 ± 53.2 (46∼223)
Mutant allele (%) (range)	50.1 ± 17.1 (33∼83)	55.7 ± 23.1 (38∼100)

TOOLS

Similar articles