Choosing the Right NGS Sequencing Instrument for Your Study

⬅️ NGS Handbook

The right sequencing instrument for your study depends on your project goal. Setting aside turnaround time and price, it essentially comes down to the numbers of reads and read length you need for your experiment. Below, we've described and compared metrics for each of the instruments available. If you’re new to high-throughput sequencing and have questions about how you should design your sequencing run, fill out our free consultation form and we'll get in touch with you to help.

Apart from some minor considerations Genohub's NGS Matching Engine makes it very easy to see what instruments fit your project best. Simply enter your specifications and instantly see services that match your output requirements.


NGS Platforms


Table 1: Comparison of all NGS Instruments


Platform Instrument Unit Reads/unit Read Length (bp) Read Type Error Type Highlight Reference
Illumina NovaSeq 6000 S4 Run 10,000,000,000 300 SR & PE substitution Launched in January 2017, Novaseq is Illumina's latest high-output instrument. The instrument is designed for research labs that can't afford the capital costs of the HiSeq X. Perhaps more important, the instrument does not have application restrictions. 12
Illumina NovaSeq 6000 S3 Run 6,600,000,000 300 SR & PE substitution NovaSeq has 4 different types of flow cells (S1-S4), each with different read outputs. 12
Illumina NovaSeq 5000/6000 S2 Run 3,300,000,000 300 SR & PE substitution 12
Illumina NovaSeq 5000/6000 S1 Run 1,600,000,000 300 SR & PE substitution 12
Illumina NextSeq 500 High-Output Run 400,000,000 300 SR & PE substitution Highest output of any desktop sequencer, this instrument is ideally suited for exome, transcriptomics, whole genome and targeted resequencing. 2
Illumina HiSeq X Lane 375,000,000 300 PE substitution Greatest throughput and number of reads compared to all other instruments, designed for human and non-human whole human genome sequencing. 1
Illumina HiSeq 3000/4000 Lane 312,500,000 300 SR & PE substitution Takes advantage of patterned flow cell technology to get more reads/lane. The HiSeq 3000 has an output of 750 Gb or 2.5B PE150 reads in 3.5 days. The HiSeq 4000 has two flow cells, so twice the output: 1.5 Tb, 5B PE150 reads in 3.5 days. 1
Illumina NextSeq 500 Mid-Output Run 130,000,000 300 PE substitution 2
Illumina HiSeq High-Output v4 Lane 250,000,000 250 SR & PE substitution Well suited for de novo and resequencing of small and large genomes, this Illumina instrument is today's sequencing workhorse. Latest v4 chemistry now allows more reads per lane and a slightly longer read length, 2x125 bp 3
Illumina HiSeq High-Output v3 Lane 186,048,000 250 SR & PE substitution v3 chemistry offers fewer reads per lane compared to v4 3
Illumina HiSeq Rapid run v4 Lane 150,000,000 500 SR & PE substitution Same as HiSeq High-output, except fewer reads 3
Illumina HiSeq Rapid Run Lane 150,696,000 300 SR & PE substitution Same as HiSeq run, except option available for faster run time, slightly less reads 3
Illumina HiScanSQ Lane 93,024,000 200 SR & PE substitution Versatility to scan miroarray and sequence on the same instrument, output slightly less than Hiseq. Discontinued by Illumina in 2014. 3
Illumina GAIIx Lane 42,075,000 300 SR & PE substitution Higher per bp cost than HiSeq, no future developement planned by Illumina. Discontinued in 2014. 3
Illumina MiSeq v3 Lane 25,000,000 600 SR & PE substitution Illumina's longest read instrument, the MiSeq offers 2x300 bp read lengths if v3 cluster chemistry is used. Read quality tends to drop on read 2 around 200-250 bases in, an issue Illumina is aware of. 3
Illumina MiniSeq High-Output Lane 25,000,000 300 SR & PE substitution Illumina's lowest output instrument, the MiniSeq, is ideal for targeted sequencing applications where a high output of reads isn't required. The instrument has an attractive $50K entry price. 11
Illumina MiSeq v2 Lane 16,000,000 250 SR & PE substitution 3
Illumina MiniSeq Mid-Output Lane 8,000,000 300 SR & PE substitution MiniSeq has a Mid-Output mode that offers 3x fewer reads than High-Out put mode. 11
Illumina MiSeq v2 Micro Lane 4,000,000 300 SR & PE substitution 4
Illumina MiSeq v2 Nano Lane 1,000,000 500 SR & PE substitution 4
Ion Proton I Chip 60,000,000 200 SR indel Ion instrument with highest throughput. Compared to MiSeq, has greater number of reads but shorter read lengths. 5
Ion PGM 318 Chip 4,000,000 400 SR indel Fast turnaround time, optimal for small genomes or targeted sequencing. Compared to MiSeq, has fewer number of reads and shorter read length. 5
Ion PGM 316 Chip 2,000,000 400 SR indel 5
Ion PGM 314 Chip 400,000 400 SR indel 5
PacBio PacBio Sequel SMRT Cell 370,000 20,000 SR indel PacBio's first desktop instrument delivers ~7x more reads than its predecessor. 13
PacBio PacBio RS II (P6) SMRT Cell 55,000 15,000 SR indel 6
Roche 454 GS FLX+ / FLX 1 PTP 700,000 700 SR indel Long read lengths make it ideal for sequencing of small genomes. 8
Roche 454 GS FLX+ / FLX 1/2 PTP 350,000 700 SR indel 8
Roche 454 GS FLX+ / FLX 1/4 PTP 125,000 700 SR indel 8
Roche 454 GS FLX+ / FLX 1/8 PTP 50,000 700 SR indel 8
Roche 454 GS FLX+ / FLX 1/16 PTP 20,000 700 SR indel 8
Roche 454 GS FLX+ / FLX 1 PTP 70,000 400 SR indel 8
SOLiD 5500xl W Lane 266,666,667 100 SR & PE A/T Bias High throughput, good for re-sequencing. Short reads make it non-ideal for de novo assembly 9
SOLiD 5500 W Lane 266,666,667 100 SR & PE A/T Bias 9
SOLiD 5500 Lane 81,500,000 100 SR & PE A/T Bias 10
SOLiD 5500xl Lane 81,500,000 100 SR & PE A/T Bias 10
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Illumina

Illumina instruments are versatile and ideal for a variety of sequencing applications, including assembly, resequencing, transcriptome, SNP detection and metagenomic studies. The HiSeq, Nextseq and GAIIx instruments are well suited for analyzing large animal or plant genomes. High level multiplexing of samples is possible when analyzing species with a smaller genome size. The HiSeq X instrument, released by Illumina in early 2014, has been been specifically designed for sequencing of whole genomes. The Illumina MiSeq outputs significantly fewer reads (Table 1), but its read lengths are significantly longer, making it ideal for small genomes, sequencing long variable domains or targeted regions within a genome. In fact, out of all the Illumina instruments, the MiSeq is best suited for amplicon-seq, 16S and other low diversity read based applications. The MiSeq Control Software (MCS) now allows users to use as little as 5% PhiX DNA spike-in to successfully sequence low-diversity samples. Other instruments, including the HiSeq and GAIIx still require at least 20-50% PhiX and are less suited for low diversity samples. The NextSeq 500, released in March of 2014, uses a two channel SBS sequencing process, likely making it even less suited for low diversity amplicons. As of 4/2014, Illumina has not performed significant validation or testing using low diversity samples on the NextSeq 500. It is not expected that the NextSeq 500 instrument will perform better than the HiSeq for these sample types. The only other major limitation of Illumina instruments compared to others in this list is their relatively short reads (2x300 is currently the longest MiSeq paired end read length).

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ION

The Ion PGM (Ion Torrent), is ideal for amplicons, small genomes or targeting of small regions within a genome. Its low throughput makes it ideal for smaller sized studies. The Ion Proton however is capable of generating significantly larger outputs (Table 1) making sequencing of transcriptome, exome and medium sized genomes possible.

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Pacific Biosystems

The PacBio RS/RS II breaks the mold of other short reads high throughput sequencing instruments by focusing on length. The reads, averaging ~4.6 kb are significantly longer than other sequencing platforms making it ideal for sequencing small genomes such as bacteria or viruses. Other advantages include its ability to sequence regions of high G/C content and determine the status of modified bases (methylation, hydroxymethylation), eliminating the need for chemical conversion during library preparation. The instrument’s low output of reads prevent it from being useful for assembly of medium to large genomes.

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Roche 454

The Roche 454 FLX+ is typically used in studies where read length is critical. These include de novo assemblies of microbial genomes, BACs and plastids. Its long read length has made it a favorite of those examining 16S variable regions and other targeted amplicon sequences. The lower output of the FLX and FLX+ instruments makes it less cost-effective for transcriptome or larger genome studies. Roche has announced that it will stop manufacturing the 454 in 2015 and end servicing in mid-2016.

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SOLiD

The SOLiD series of instruments are high throughput, generating a large number of short reads. De novo sequencing, differential transcript expression and resequencing are all viable applications of the SOLiD platform. The weakness of the platform is its short reads making assembly very difficult.

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