⬅️ 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
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NGS Platforms
Table 1: Comparison of all NGS Instruments
| Platform |
Instrument |
Unit |
Reads/Unit* |
Max Read Length |
Read Type |
Error Type |
Highlight |
Reference |
| Illumina |
GAIIx
|
Lane |
37,500,000 |
2x150bp |
SR & PE |
Substitution |
Higher per bp cost than HiSeq, no future development planned by Illumina. Discontinued in 2014. |
Reference |
| Illumina |
HiScanSQ
|
Lane |
93,750,000 |
2x100bp |
SR & PE |
Substitution |
Versatility to scan microarray and sequence on the same instrument, output slightly less than HiSeq. Discontinued by Illumina in 2014. |
Reference |
| Illumina |
HiSeq 3000/4000
|
Lane |
312,500,000 |
1x300bp, 2x150bp |
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. |
Reference |
| Illumina |
HiSeq High-Output v3
|
Lane |
187,500,000 |
2x150bp |
SR & PE |
Substitution |
v3 chemistry offers fewer reads per lane compared to v4 |
Reference |
| Illumina |
HiSeq High-Output v4
|
Lane |
250,000,000 |
1x250bp, 2x125bp |
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 |
Reference |
| Illumina |
HiSeq Rapid Run
|
Lane |
150,000,000 |
1x500bp, 2x250bp |
SR & PE |
Substitution |
Same as HiSeq run, except option available for faster run time, slightly less reads |
Reference |
| Illumina |
HiSeq X
|
Lane |
375,000,000 |
2x150bp |
PE |
Substitution |
Greatest throughput and number of reads compared to all other instruments, designed for human and non-human whole human genome seqencing. |
Reference |
| Illumina |
MiSeq
|
Lane |
5,000,000 |
2x250bp |
SR & PE |
Substitution |
|
Reference |
| Illumina |
MiSeq v2
|
Lane |
17,000,000 |
2x250bp |
SR & PE |
Substitution |
|
Reference |
| Illumina |
MiSeq v2 Micro
|
Lane |
4,000,000 |
2x150bp |
SR & PE |
Substitution |
|
Reference |
| Illumina |
MiSeq v2 Nano
|
Lane |
1,000,000 |
2x250bp |
SR & PE |
Substitution |
|
Reference |
| Illumina |
MiSeq v3
|
Lane |
25,000,000 |
2x300bp |
SR & PE |
Substitution |
Illumina's longest read instrument, the MiSeq offer 2x300 bp read lengths if v3 cluster chemistry is used. Read quality tends to drop on read 2 around 200-250 bases in, as issue Illumina is aware of. |
Reference |
| Illumina |
MiniSeq High-Output
|
Lane |
25,000,000 |
2x150bp |
SR & PE |
Substiution |
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. |
Reference |
| Illumina |
MiniSeq Mid-Output
|
Lane |
8,000,000 |
2x150bp |
SR & PE |
Substitution |
MiniSeq has a Mid-Output mode that offers 3x fewer reads than High-Out put mode. |
Reference |
| Illumina |
MiniSeq Rapid
|
Lane |
20,000,000 |
1x100 |
SR |
Substitution |
|
Reference |
| Illumina |
NextSeq 1000/2000 P1
|
Run |
100,000,000 |
1x300bp, 2x150bp |
SR & PE |
Substitution |
|
Reference |
| Illumina |
NextSeq 1000/2000 P2
|
Run |
400,000,000 |
1x300bp, 2x150bp |
SR & PE |
Substitution |
|
Reference |
| Illumina |
NextSeq 1000/2000 P3
|
Run |
1,200,000,000 |
1x300bp, 2x150bp |
SR & PE |
Substitution |
|
Reference |
| Illumina |
NextSeq 500 High-Output
|
Run |
400,000,000 |
1x300bp, 2x150bp |
SR & PE |
Substitution |
Highest output of any desktop sequencer, this instrument is ideally suited for exome, transcriptomics, whole genome and targeted resequencing. |
Reference |
| Illumina |
NextSeq 500 Mid-Output
|
Run |
130,000,000 |
2x150bp |
PE |
Substitution |
|
Reference |
| Illumina |
NextSeq 500 v2 High-Output
|
Run |
400,000,000 |
1x300bp, 2x150bp |
SR & PE |
Substitution |
Highest output of any desktop sequencer, this instrument is ideally suited for exome, transcriptomics, whole genome and targeted resequencing. |
Reference |
| Illumina |
NextSeq 500 v2 Mid-Output
|
Run |
130,000,000 |
2x150bp |
PE |
Substitution |
|
Reference |
| Illumina |
NovaSeq - S1
|
Lane |
800,000,000 |
1x300bp, 2x150bp |
SR & PE |
Substitution |
|
Reference |
| Illumina |
NovaSeq - S2
|
Lane |
1,650,000,000 |
1x300bp, 2x150bp |
SR & PE |
Substitution |
|
Reference |
| Illumina |
NovaSeq - S4
|
Lane |
2,500,000,000 |
1x300bp, 2x150bp |
SR & PE |
Substitution |
Launced 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. |
Reference |
| Illumina |
NovaSeq - SP
|
Lane |
400,000,000 |
1x500bp, 2x250bp |
SR & PE |
Substitution |
|
Reference |
| Illumina |
NovaSeq X - 1.5B
|
Lane |
200,000,000 |
2x50bp to 2x150bp |
PE |
|
High output per flow cell. Great for large whole genome, whole exome and transcriptomic projects. |
Reference |
| Illumina |
NovaSeq X - 10B
|
Lane |
1,250,000,000 |
2x50bp to 2x150bp |
PE |
|
High output per flow cell. Great for large whole genome, whole exome and transcriptomic projects. |
Reference |
| Illumina |
NovaSeq X - 25B
|
Lane |
3,250,000,000 |
2x50bp to 2x150bp |
PE |
|
High output per flow cell. Great for large whole genome, whole exome and transcriptomic projects. |
Reference |
| Illumina |
NovaSeq X Plus - 1.5B
|
Lane |
206,250,000 |
2x50bp to 2x150bp |
PE |
|
High output per flow cell. Great for whole genome, whole exome and transcriptomics projects. |
Reference |
| Illumina |
NovaSeq X Plus - 10B
|
Lane |
1,250,000,000 |
2x50bp to 2x150bp |
PE |
|
High output per flow cell. Great for whole genome, whole exome and transcriptomics projects. |
Reference |
| Illumina |
NovaSeq X Plus - 25B
|
Lane |
3,331,250,000 |
2x50bp to 2x150bp |
PE |
|
High output per flow cell. Great for whole genome, whole exome and transcriptomics projects. |
Reference |
| Illumina |
iSeq 100
|
Lane |
4,000,000 |
2x150bp |
SR & PE |
Substitution |
|
Reference |
| PacBio |
PacBio RS
|
SMRT Cell |
22,000 |
4500bp |
SR |
Indel |
|
Reference |
| PacBio |
PacBio RS II
|
SMRT Cell |
47,000 |
4600bp |
SR |
Indel |
|
Reference |
| PacBio |
PacBio RS II (P4)
|
SMRT Cell |
47,000 |
5500bp |
SR |
Indel |
|
Reference |
| PacBio |
PacBio RS II (P5)
|
SMRT Cell |
47,000 |
8500bp |
SR |
Indel |
|
Reference |
| PacBio |
PacBio RS II (P6)
|
SMRT Cell |
47,000 |
20000bp |
SR |
Indel |
|
Reference |
| PacBio |
PacBio Sequel
|
SMRT Cell |
187,500 |
20000bp |
SR |
Indel |
PacBio's first desktop instrument delivers ~7x more reads than its predecessor. |
Reference |
| PacBio |
PacBio Sequel II
|
SMRT Cell |
1,500,000 |
20000bp |
SR |
Indel |
|
Reference |
| Oxford Nanopore |
Flongle with kit 10/11 chemistry
|
Flow cell |
None |
typically 6-20 Kbp |
SR |
Indel & substitution |
Adapter that can be attached to the MinION and GridION devices for smaller, rapid tests. Especially useful and cost-effective at $90 per flow cell for running single samples or pilots. |
Reference |
| Oxford Nanopore |
Flongle with kit 12 chemistry
|
Flow cell |
None |
typically 6-20 Kbp |
SR |
Indel & substitution |
Adapter that can be attached to the MinION and GridION devices for smaller, rapid tests. Especially useful and cost-effective at $90 per flow cell for running single samples or pilots. |
Reference |
| Oxford Nanopore |
GridION with kit 10/11 chemistry
|
Flow cell |
None |
typically 6-20 Kbp |
SR |
Indel & substitution |
Very flexible, as it can run 1-5 Flongle or MinION flow cells concurrently as independent runs with an integrated data processing and analysis unit. Best for larger whole genome or transcriptome projects. |
Reference |
| Oxford Nanopore |
GridION with kit 12 chemistry
|
Flow cell |
None |
typically 6-20 Kbp |
SR |
Indel & substitution |
Very flexible, as it can run 1-5 Flongle or MinION flow cells concurrently as independent runs with an integrated data processing and analysis unit. Best for larger whole genome or transcriptome projects. |
Reference |
| Oxford Nanopore |
MinION with kit 10/11 chemistry
|
Flow cell |
None |
typically 6-20 Kbp |
SR |
Indel & substitution |
Portable, pocket-sized and useful for when there’s a need to run real-time sequencing in the field. The low cost makes it a viable sequencing option for almost any lab for metagenomes, targeted sequencing and smaller genomes and transcriptomes. |
Reference |
| Oxford Nanopore |
MinION with kit 12 chemistry
|
Flow cell |
None |
typically 6-20 Kbp |
SR |
Indel & substitution |
Portable, pocket-sized and useful for when there’s a need to run real-time sequencing in the field. The low cost makes it a viable sequencing option for almost any lab for metagenomes, targeted sequencing and smaller genomes and transcriptomes. |
Reference |
| Oxford Nanopore |
PromethION with kit 10/11 chemistry
|
Flow cell |
None |
typically 6-20 Kbp |
SR |
Indel & substitution |
Largest device that can run 1-48 independent flow cells on-demand and analyze the data in real time. Can generate Tb of data and is best used for very high-through sequencing for large whole genomes and transcriptomes or population-scale sequencing. |
Reference |
| Oxford Nanopore |
PromethION with kit 12 chemistry
|
Flow cell |
None |
typically 6-20 Kbp |
SR |
Indel & substitution |
Largest device that can run 1-48 independent flow cells on-demand and analyze the data in real time. Can generate Tb of data and is best used for very high-through sequencing for large whole genomes and transcriptomes or population-scale sequencing. |
Reference |
| Ion Torrent |
PGM 314 Chip
|
Chip |
400,000 |
400bp |
SR |
Indel |
|
Reference |
| Ion Torrent |
PGM 316 Chip
|
Chip |
2,000,000 |
400bp |
SR |
Indel |
|
Reference |
| Ion Torrent |
PGM 318 Chip
|
Chip |
4,000,000 |
400bp |
SR |
Indel |
Fast turnaround time, optimal for small genomes or targeted sequencing. Compared to MiSeq, has fewer number of reads and shorter read length. |
Reference |
| Ion Torrent |
Proton I Chip
|
Chip |
60,000,000 |
200bp |
SR |
Indel |
Ion instrument with highest throughput. Compared to MiSeq, has greater number of reads but shorter read lengths. |
Reference |
| Ion Torrent |
S5/S5 XL 520 Chip
|
Chip |
5,000,000 |
400bp |
SR |
Indel |
|
Reference |
| Ion Torrent |
S5/S5 XL 530 Chip
|
Chip |
20,000,000 |
400bp |
SR |
Indel |
|
Reference |
| Ion Torrent |
S5/S5 XL 540 Chip
|
Chip |
80,000,000 |
200bp |
SR |
Indel |
|
Reference |
| Roche454 |
GS FLX 1 PTP
|
1 PTP |
700,000 |
450bp |
SR |
Indel |
Long read lengths make it ideal for sequencing of small genomes. |
|
| Roche454 |
GS FLX 1/16 PTP
|
1/16 PTP |
20,000 |
450bp |
SR |
Indel |
|
|
| Roche454 |
GS FLX 1/2 PTP
|
1/2 PTP |
350,000 |
450bp |
SR |
Indel |
|
|
| Roche454 |
GS FLX 1/4 PTP
|
1/4 PTP |
125,000 |
450bp |
SR |
Indel |
|
|
| Roche454 |
GS FLX 1/8 PTP
|
1/8 PTP |
50,000 |
450bp |
SR |
Indel |
|
|
| Roche454 |
GS FLX+ 1 PTP
|
1 PTP |
700,000 |
700bp |
SR |
Indel |
Long read lengths make it ideal for sequencing of small genomes. |
|
| Roche454 |
GS FLX+ 1/16 PTP
|
1/16 PTP |
20,000 |
700bp |
SR |
Indel |
|
|
| Roche454 |
GS FLX+ 1/2 PTP
|
1/2 PTP |
350,000 |
700bp |
SR |
Indel |
|
|
| Roche454 |
GS FLX+ 1/4 PTP
|
1/4 PTP |
125,000 |
700bp |
SR |
Indel |
|
|
| Roche454 |
GS FLX+ 1/8 PTP
|
1/8 PTP |
50,000 |
700bp |
SR |
Indel |
|
|
| Roche454 |
GS Junior 1 PTP
|
1 PTP |
70,000 |
400bp |
SR |
Indel |
|
|
| BGI |
DNBSEQ-G400 FAST
|
Lane |
275,000,000 |
2x150bp |
SR & PE |
|
|
Reference |
| BGI |
DNBSEQ-G400 FCL
|
Lane |
450,000,000 |
1x400bp, 2x200bp |
SR & PE |
|
|
Reference |
| BGI |
DNBSEQ-G400 FCS
|
Lane |
275,000,000 |
2x150bp |
SR & PE |
|
|
Reference |
| BGI |
DNBSEQ-G50 FCL
|
Lane |
500,000,000 |
2x150bp |
SR & PE |
|
|
Reference |
| BGI |
DNBSEQ-G50 FCS
|
Lane |
100,000,000 |
2x150bp |
SR & PE |
|
|
Reference |
| BGI |
DNBSEQ-T7
|
Lane |
5,000,000,000 |
2x150bp |
PE |
|
|
Reference |
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).
Oxford Nanopore
Oxford Nanopore instruments are ideal for sequencing short to ultra-long fragments of native DNA or
RNA and can be used for various applications. The main limitation of Oxford Nanopore sequencing is
the comparatively lower read accuracy compared to short read sequencing. The relatively higher
insertion and election error rates make Nanopore sequencing less optimal for single nucleotide
variation/polymorphism (SNV or SNP) detection, unless you can obtain high coverage. Oxford Nanopore
has improved on this limitation with the latest R10.3 chemistry, which allows for higher throughput
and capture and improved raw accuracy to support enhanced variant calling.
Starting from the smallest instrument, the Flongle is an adapter for the MinION or GridION platforms
and is best for amplicon sequencing, small panel/targeted sequencing, quality testing for a pilot
run before a larger sequencing experiment, and running single samples on demand rather than
multiplexing. The MinION is a larger, portable instrument and is best used for whole genomes, whole
exomes, metagenomes, whole transcriptomes (cDNA), smaller transcriptomes (direct RNA), targeted
sequencing and multiplexing for smaller samples. The GridION is a benchtop instrument designed to
run up to five MinION or Flongle Flow Cells, each of which can be independently controlled in real
time. The GridION is ideal for larger genomes, whole transcriptomes (direct RNA or cDNA) and a
higher number of samples. The PromethION is the largest instrument and is used for high-coverage
sequencing. At this level, each flow cell delivers the lowest price per Gb for nanopore sequencing,
making it ideal for larger genomes, population-scale experiments, highly multiplexed small genomes
or targeted regions and whole transcriptomes (direct RNA or cDNA).
Ion Torrent
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.
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.
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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