2016 NGS Field Guide – Table 1 – Instrument Use Grades

Table 1a – 2014. Utility of 2nd and 3rd generation DNA sequencing platforms for de novo assemblies of different templates.  Table assumes Illumina, Ion Torrent, and Oxford Nanopore achieve stated goals (independent of stated time-lines). Initial letter indicates the author’s opinion of the overall utility (grade) for a platform for a specific application. Utility grades combine data characteristics (amount, quality, length), cost of data, and ease of assembling the data into the final desired product. Major considerations for utility grades are noted.

Platform – instrument Application: De novo assemblies
BACs, plastids, & microbial genomes Transcriptome Plant & animal genomes
MiSeq A – good, need to multiplex for best economics A/B –expensive for rare transcripts (compared to HiSeq), but reads are longer for better assembly B – expensive relative to HiSeq, but additional read length can be valuable
NextSeq 500 A/B – need to multiplex for best economics; assembly more challenging than MiSeq long reads A/B – more expensive than HiSeq A/B – more expensive than HiSeq
HiSeq 2500 – high output B – more data than needed unless highly indexed; assembly more challenging than MiSeq long reads A – primary data type in most current projects A – primary data type in most current projects; requires mate-pair libraries
HiSeq 2500 – rapid run B – more data than needed unless highly indexed; assembly more challenging than longest MiSeq reads A – good alternative to HiSeq high output A – slightly more expensive than HiSeq2000, but gives increased read length
HiSeq X

(projected)

C – much more data than needed unless highly indexed A – not supported initially, but should be a good alternative to HiSeq 2500 A – What this machine was designed to do
Ion Torrent – PGM 314 B – OK, lowest experimental cost but reads are shorter & more expensive than Illumina D – OK, but reads are much more expensive than Illumina D – cost prohibitive
Ion Torrent – PGM 318 B – good, but shorter reads and less data with more errors than MiSeq C – OK, but reads are more expensive than Illumina D – cost prohibitive
Ion Torrent Proton I B – more data than needed unless indexed; assembly more challenging than 454 or Illumina B – assembly currently more challenging than Illumina or 454 B – expensive relative to HiSeq or Proton II/III
PacBio – RS B/A – good, especially for hybrid assemblies, but expensive to use solo due to high error rates B/D – good for hybrid assemblies; too expensive and inaccurate for solo use B/D – good for hybrid assemblies & scaffolding (mixed platform strategy); cost prohibitive for solo use
454 – GS Jr. C – OK but expensive D – cost prohibitive D – cost prohibitive
454 – FLX+ B – good, need to multiplex to be economical C – good but expensive, libraries usually normalized, not best for short RNAs C – OK as part of a mixed platform strategy, prohibitive to use alone
Ion Torrent Proton II (projected) B – more data than needed unless highly indexed; assembly more challenging than 454 or Illumina B/A – assembly currently more challenging than Illumina or 454 B – should be similar to NextSeq or HiSeq
Ion Torrent Proton III

(forecast)

C – more data than needed unless highly indexed B/A – need assembly pipelines B/A – cost per MB could make it the best
minION (forecast) B – more expensive than GridION B – more expensive than GridION C/D – cost prohibitive except for scaffolding
GridION (forecast) B/A – great for scaffolding, will likely need high coverage or to combine with short reads with lower error rates B/A – great for defining full-length transcripts, will need to combine with short reads with lower error rate until error rates are reduced B/A – great for scaffolding, will need to combine with short reads with lower error rate until error rates are reduced
SOLiD – 5500

 

C – too much data unless highly indexed; assembly more challenging than 454, Ion Torrent or Illumina C/D – short reads make assembly challenging or impossible C/D – short reads make assembly challenging or impossible

Table 1b – 2014. Utility of 2nd and 3rd generation DNA sequencing platforms for resequencing applications. (2014 information)Table assumes Illumina, Ion Torrent, and Oxford Nanopore achieve stated goals (independent of stated time-lines). Initial letter indicates the author’s opinion of the overall utility (grade) for a platform for a specific application. Utility grades combine data characteristics (amount, quality, length), cost of data, and ease of assembling the data into the final desired product. Major considerations for utility grades are noted.

Platform – instrument Application: Resequencing
Targeted loci Transcript counting Genome resequencing
MiSeq A/B – best for longest reads, but at higher cost per read than HiSeq C – more expensive than HiSeq or SOLiD or Proton II+ C – expensive for large genomes
NextSeq 500 A – slightly more expensive than HiSeq B – more expensive than HiSeq B – more expensive than HiSeq
HiSeq 2500 – high output A – primary data type in many current projects; best for many loci A – primary data type in many current projects A – primary data type in many current projects
HiSeq 2500 – rapid run A – faster path to leading data type A/B – likely to be more expensive than with standard flow cell A – faster path to leading data type
HiSeq X

(projected)

A – should be lower cost than HiSeq A – should be lower cost than HiSeq A – what this was designed for
Ion Torrent – PGM 314 C – OK but expensive, need to limit loci D – cost prohibitive D – cost prohibitive
Ion Torrent – PGM 318 B – good, less data per run than MiSeq C – more expensive than Illumina, SOLiD or Proton C – expensive for large genomes
Ion Torrent Proton I B/A – similar to MiSeq, but different error profile will inhibit switching B – more expensive than Illumina or SOLiD B – expensive relative to HiSeq or Proton II+
PacBio – RS C – expensive but can sequence long and difficult regions D – cost prohibitive C/D – cost prohibitive except for structural variants
454 – GS Jr. B/C – good but expensive, need to limit loci D – cost prohibitive D – cost prohibitive for large genomes
454 – FLX+ B – good but expensive, should limit loci D – cost prohibitive D – cost prohibitive for large genomes
SOLiD – 5500xl B – harder to assemble than Illumina A/B – used much less than HiSeq A/B – used much less than HiSeq
Ion Torrent Proton II (projected) B/A – similar to HiSeq, but different error profile will inhibit switching B/A – new informatics pipelines needed B/A – will be attractive sometimes, especially to verify assemblies of Illumina data
Ion Torrent Proton III

(forecast)

A/B – costs projected to be better than HiSeq; error profile different than Illumina A/B – new informatics pipelines needed A – supposed to set new pricing standard, could become leading shorter-read platform
minION (forecast) C? – error profile may make this less desirable D – cost prohibitive C/D? – expensive for large genomes
GridION (forecast) B? – error profile may make this less desirable B? – error profile may make this less desirable B? – error profile may make this less desirable

Table 1c – 2014. Utility of 2nd and 3rd generation DNA sequencing platforms for various applications. Table assumes Illumina, Ion Torrent, and Oxford Nanopore achieve stated goals (independent of stated time-lines). Initial letter indicates the author’s opinion of the overall utility (grade) for a platform for a specific application. Utility grades combine data characteristics (amount, quality, length), cost of data, and ease of assembling the data into the final desired product. Major considerations for utility grades are noted.

Platform – instrument Various applications
Metagenomics1 Mutation Detection2 Other limitations
MiSeq A – beware phase* B – more expensive than HiSeq, SOLiD, or Proton II+ No inexpensive kits
NextSeq 500 B – good, limited by short reads, beware phase* A/B – more expensive than HiSeq New chemistry with unknown error profile
HiSeq 2500 – high output B – OK, limited by short reads, beware phase* A – primary data type in many current projects Long time to obtain data (weeks to months)
HiSeq 2500 – rapid run B – good, limited by short reads, beware phase* A – primary data type in many current projects Fewer reads per lane than high output
HiSeq X (projected) B – good, limited by short reads, beware phase* A – should become primary data type Single machines not available anytime soon; Limited applications supported initially
Ion Torrent – PGM 314 B – limited by read number and cost D – cost prohibitive Escalating costs with increased read length
Ion Torrent – PGM 318 B – possibly replacing 454; fewer phase issues C – more expensive than MiSeq, HiSeq, SOLiD, or Proton Escalating costs with increased read length
Ion Torrent Proton C – shorter read-length than 454 or MiSeq, longer reads than HiSeq B – more expensive than HiSeq or SOLiD; different error profile Will 400 base reads become available? Reductions in kit costs?
PacBio – RS C/D – high costs, but long reads allow scaffolding in hybrid sequencing strategies F – cost prohibitive & insufficient accuracy When will new applications be available?
454 – GS Jr. B/C – good but costs limit sample number & depth D – cost prohibitive Customer unhappiness; end of life = 2016
454 – FLX+ B – good but expensive, long reads maximize data per read C – expensive, good for identifying clusters Reliability issues; Customer unhappiness; end of life = 2016
Ion Torrent Proton II (projected) B/A – shorter read-length than 454 or MiSeq, longer reads than HiSeq B/A – similar in cost to HiSeq & SOLiD; different error profile ≥400 base reads? Will they finally catch Illumina?
Ion Torrent Proton III

(forecast)

B/A – shorter read-length than 454 or MiSeq, longer reads than HiSeq B – projected to be cost leader; different error profile ≥400 base reads? Will they finally beat Illumina before the nanopores come on-line?
minION (forecast) B – excellent for environmental sample sequencing; enrichment techniques will need to be developed; field portable; limited by accuracy D? – accuracy is likely limiting for this application Early access program has been initiated, but when these will become available generally and at what cost is still unknown
GridION (forecast) A/B – excellent for environmental sample sequencing; enrichment techniques will be helpful; limited by accuracy ? – accuracy is likely limiting for this application When will this be available? How much will the nodes and reagents cost?
SOLiD – 5500xl D – limited by short reads B – frequent data type in many current projects Limited future

1Metagenomics – characterization of 16S sequences within and among microbial communities, primarily via sequencing amplicons; see genome assembly scores for metagenomics.

2Mutation Detection – identification of rare sequence variants

*Illumina instruments require a mixture of different base signals among clusters during each cycle, thus amplicon sequencing requires strategies to offset the beginning bases of amplicons or use of custom sequencing primers