Background
NGS technology
NGS applications
Next-generation platforms
Roche
Roche | Illumina | Thermo Fisher | Pacific Biosciences | Oxford Nanopore | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
GS Junior | GS FLX + | MiniSeq | MiSeq | NextSeq | HiSeq/HiSeqX | NovaSeq | SOLID 5500W | Ion PGM | Ion Proton | Ion S5 | RS II | Sequel | MinION | GridION/PromethION | |
Max output | 35 Mb | 700 Mb | 7.5 Gb | 15 Gb | 120 Gb | 1500/1800 Gb | 6000 Gb | 320 Gb | 1–2 Gb | 10 Gb | 10–15 Gb | 1 Gb | 10 Gb | 10–20 Gb | 50–100 Gb/ Tb scale |
Max reads/run | 0.1 M | 1 M | 50 M (PE) | 50 M (PE) | 800 M (PE) | 3 G (PE) | 20 G | 1.4 G | 5.5 M | 60–80 M | 3–80 M | 50 Kb | 500 Kb | 2–4 M | 14–1000 M |
Max read length | 400 b | 1000 b | 2 × 150 b | 2 × 300 b | 2 × 150 b | 2 × 150 b | 2 × 150 b | 50 b | 400 b | 200 b | 600 b | 15 Kb (mean read lenght) | 15 Kb (mean read lenght) | 300 K | 300 K |
Accuracy (max) | 99% at 400 b | 99.9% at 15× coverage E. coli
| 99.9% for more than 80–85% of bases | 99.9% for more than 75–90% of bases | 99.9% for more than 75–80% of bases | 99% for more than 75–85% of bases | 99.9% for more than 75–85% of bases | > 99% | > 99% | > 99% | 99% (expected) | 99% | 99% | 96% | 96% |
Run time | 10 h | 10–23 h | 7–24 h | 4–56 h | 12–30 h | 1–3.5/<3 days | 19–40 h | 10 days | 2–7 h | 2–4 h | 2.5 h | 0.5–10 h | 0.5–10 h | 2 min–48h | 2 min–48 h |
DNA/RNA inputa
| 500 ng | 200 ng | 1–200 ng | 1–100 ng | 50 ng–1 µg | 50 ng–1 µg | 100 ng–1 µg | 10 ng–5 µg | 1–100 ng | 1–100 ng | 1–100 ng | 10 ng–1 µg | 10 ng–1 µg | 200 ng | 200 ng |
Max prep time/ Hands-on | 24 h | 24 h | 1 day/ 4 h | 7 h | 9 h/ 6 h | 2.5 days | 2.5 days | 8 h | 10 h/ 4.5 h | 10 h/ 45 min | 45 min hands-on | 6 h/ 4 h | 6 h/ 4 h | 10 min (1D) | 10 min (1D) |
Key applications | A | B | C | D | E | F/G | F | H | I | L | M | N | O | P | P |
Illumina-solexa
Ion Torrent Thermo Fischer
SOLiD
Pacific Biosciences
Oxford Nanopore technologies
Qiagen GeneReader
Bionano technologies
NGS platforms comparison
Colorectal cancer (CRC)
CRC: main features
Molecular genetics of CRC
Connections between CRC and gut microbiota
CRC therapy
NGS in CRC
Mutation analysis of actionable genes
Samples | NGS platform | # of analyzed genes/assay | Mutant genes analyzed | Therapy | Ref. |
---|---|---|---|---|---|
320 mCRC | 454 GS FLX | 9 Targeted-seq (KRAS, NRAS, EGFR, BRAF, PTEN, PI3KCA, AKT, TP53, CTNNB1) | K/NRAS, BRAF, PI3KCA, TP53, PTEN, EGFR, AKT, CTNNB1 | Panitumumab | Peeters et al. [75] |
468 CRC (77 KRAS analyzed) | GAIIx Illumina | 1321 Targeted-seq (SureSelect Agilent) | KRAS | Anti-EGFR | Kothari et al. [76] |
182 mCRC KRAS ex2 wt | Ion Torrent | 22 Targeted-seq (AmpliSeq colon and lung cancer panel) | KRAS (29/182pts) frequently mutant: TP53, RAS, PI3KCA, BRAF | Folfiri/Cetuximab | Ciardiello et al. [77] |
91 CRC | Ion Torrent | 22 | KRAS, TP53, APC, FBXW7, PI3KCA, BRAF, CTNNB1, ERBB2, SMAD4 | Anti-EGFR | Bai et al. [78] |
188 mCRC | 454 GS Junior | 2 Targeted-seq (KRAS, NRAS) | KRAS, NRAS | Anti-EGFR | Harlé et al. [79] |
1054 stage III CRC | Ion Torrent | 22 | KRAS, NRAS, BRAF | Folfox/cetuximab | Taieb et al. [80] |
63 Iranian CRC | Ion Torrent | 15 Targeted-seq (AMER1, APC, ARID1A, BRAF, FBXW7, KRAS, MSH3, MSH6, NRAS, PIK3CA, SMAD4, SOX9, TCF7L2, TGFBR2 and TP53) | MSH3, MSH6, AMER1, APC, BRAF, KRAS, PI3KCA, TGFBR2A, SMAD4, SOX9, TCF7L2, TP53 | Suggested informed genetic diagnosis and personalized strategies | Ashktorab et al. [81] |
Illumina | 20 Targeted-seq (ACVR2A, AMER1, APC, ARID1A, BRAF, FBXW7, KRAS, MSH2, MSH3, MSH6, NRAS, PIK3CA, POLE, PTEN, SMAD2, SMAD4, SOX9, TCF7L2, TGFBR2 and TP53) | ||||
138 stage IV CRC | FoundationOne method | 315 cancer related genes + introns of 28 frequently rearranged genes | RAS, RAF, ERBB2, AKT, PI3KCA, PTEN, MET, POLE | Anti-EGFR, anti-HER2, anti-PD1 | Gong et al. [82] |
7 CRC cell lines 25 CRC | Illumina MiSeq | 48 Targeted-seq (TruSeq Amplicon Cancer) | ATM, E-cadherin | Anti-EGFR | Geiβler et al. [83] |
3 mCRC positive for NTRK1 rearrangement | Illumina MiSeq | 5 gene rearrangements (NTRK1/2/3, ROS, ALK) | NTRK1 | Entrectinib | Lee et al. [84] |
54 mCRCs + liver metastasis KRAS ex2 wt | Ion Torrent | 22 | KRAS, NRAS, TP53, PI3KCA, FBXW7, PTEN | Anti-EGFR | Adua et al. [85] |
53 KRAS ex2 WT mCRC | Ion Torrent | 20 Targeted-seq (EGFR, KRAS, HRAS, NRAS, BRAF, PIK3CA, AKT1, PTEN, HER2, HER4, TP53) | KRAS, NRAS, BRAF | Anti-EGFR | Hsu et al. [86] |
Novel mutations or less frequently mutant genes
Samples | NGS | # analyzed genes | Novel mutations/less frequently mutant genes | Ref. |
---|---|---|---|---|
60 pairs normal/CRC | GAIIx Illumina | 183 Targeted-seq (custom panel) | 166-point mutations 25 indels | Han et al. [90] |
4 mCRC | Illumina HiSeq | Whole genome (NEBNext DNA New England) | INPPL1 p.E567G | Shanmugam et al. [91] |
77 CRC | Ion Torrent | 22 Targeted-seq (ampliseq colon and lung) | ALK p.L1196M/AKT1, STK11, ERBB2, ERBB4, MAP2K1, NOTCH1 | Malapelle et al. [92] |
14 HNPCC, 12 EC, 2 LS | Illumina HiSeq | 22 Targeted-seq (MMR custom panel) | POLD2, EXO1 | Talseth-Palmer et al. [93] |
224 CRC | Illumina HiSeq | 341/410 cancer associated genes (Msk-impact) | POLE p.P286R | Stadler et al. [94] |
9643 mCRC | Ion Torrent | 50 Targeted-seq (Ampliseq cancer hotspot) | Non-V600 BRAF | Jones et al. [95] |
NGS transcriptomics analysis
Samples | NGS | Application | Interesting genes | Ref. |
---|---|---|---|---|
15 CRCs | Illumina HiSeq (3′-seq library) | APA changes | PPIE, DMKN, PDXK | Morris et al. [96] |
3 CRC cell lines | Roche GS Junior (primers framing CD44 variable region) | CD44 ASP | CD44 CRC specific isoforms | Bànky et al. [97] |
1 stage III CRC patient (distant, adjacent mucosa and cancer) | Illumina GAIIx (TruSeq RNA) | RNA-seq (novel and fusion transcripts, alternative splicings) | List of 14 genes with cancer-associated differential-splicing, PTGFRN-NOTCH2 fusion product | Wu et al. [98] |
70 CRCs/normal pairs | Illumina HiSeq (TruSeq RNA) | RNA-seq | RSPO2/3 fusion transcript | Seshagiri et al. [99] |
20 CRC cell lines | Illumina (ChIP-seq) | RNA-seq (genes involved in irinotecan resistance) | 20 top-genes CDC20, CTNNAL1, FZD7, CITED2, ABR, ARHGEF7 and RNMT validated by qPCR | Li et al. [100] |
21 CRC cell lines | Illumina (ChIP-seq) | RNA-seq (genes involved in oxaliplatin resistance) | 58 top-genes 15 further validated by qPCR (e.g. HNF1A, NOTCH1, FZD5, KCND1, FDZ2) | Li et al. [101] |
175 CRCs/normal tissue pairs | Illumina HiSeq (TruSeq) | RNA_seq (CRC vs. normal differentially expressed genes) | 1138 up-regulated 695 down-regulated | Slattery et al. [102] |
HCT116 CRC cell line | Illumina HiSeq | RNA-seq (SNP profiles from single cell and bulk CRC) | Genes in WNT, PI3KCA, p53, TGF-β, MMR pathways, fusion transcripts | Chen et al. [103] |
5 paired primary, mCRC, normal colon and liver tissues | Illumina HiSeq | RNA-seq (comparative transcriptome analysis) | Similar expression profiling in CRC and metastasis, RNF43-SUPT4H1 fusion transcript frequent in primary CRCs | Lee et al. [104] |
217 CRCs/normal mucosa pairs | Illumina HiSeq (TruSeq) | RNA-seq (oncogenes, tumor suppressors’ differential expression) | 22 tumor suppressor genes and 27 oncogenes significantly differentially expressed in CRC vs. normal mucosa | Slattery et al. [105] |