⬅️ 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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