Clinical applications of next-generation sequencing in colorectal cancers

Table 1 The next-generation sequencing platforms for cancer genome analysis

NGS types	Whole genome sequencing	Exome sequencing	Epigenome sequencing1	RNA-seq
Source	Genomic DNA	Genomic DNA (targeted)	Genomic DNA (targeted)	RNA
Alteration types	Point mutations and indels, rearrangements2, DNA copy number changes	Point mutations and indels	DNA methylation and posttranscriptional histone modifications	Gene fusions3, alternative splicing events, point mutations and indels

¹Epigenome sequencing can be classified into chromatin immunoprecipitation (ChIP)-based methods to detect genomic domains with epigenetic modifications[80] and more direct DNA methylation sequencing such as bisulfite-sequencing[59,60];

²From whole genome sequencing data, the genomic rearrangements and DNA copy number changes are generally detected by paired-end mapping[81] and read-depth based methods[82], respectively;

³Gene fusions can also be identified by paired-end, high-coverage whole genome sequencing, but the transcription-related events such as exon skipping or other alternative splicing events can only be identified by RNA-seq. NGS: Next-generation sequencing.

Table 2 The list of next-generation sequencing-based studies of colorectal cancer genomes

Ref.	NGS types	Major findings	Alteration types and software used
Bass et al[16]	WGS (9 pairs; tumor-matched normal)	Oncogenic fusion (VTI1A-TCG7L2)	Point mutations (MuTect[83])
			Indels (Indelocator)1
			Rearrangements (dRanger)1
TCGA consortium	WGS (97 pairs; low-pass)	See main text	Point mutations (MuTect)
	RNA-seq (218 tumors)		Recurrent mutations (MutSig)1
	Exome-seq (254 pairs)		DNA copy numbers (BIC-seq[82])
			Rearrangements (BreakDancer[81])
Timmermann et al[84]	Exome-seq (2 pairs, one MSI-H and one MSS)	Comparison of mutation spectrum between MSI-H and MSS CRC genomes	Point mutation and indel (Vendor-provided GS reference mapper, Roche)
Zhou et al[85]	Exome-seq (1 series: normal-adenoma-adenocarcinoma)	Comparison of benign and malignant CRC genomes in the same patient	Point mutation and indel (Samtools[86])
Kloosterman et al[73]	WGS (4 pairs; primary-metastasis-matched normals)Targeted 1300 genes (4 pairs)	Comparison of primary or metastatic CRC genomes	Chromothripsis and mutations (Burrow-Wheeler aligner[87] based in-house tools)
Brannon et al[88] (Proceedings)	Targeted 230 genes (50 pairs: primary-metastasis-matched normals)	Comparison of primary or metastatic CRC genomes	IMPACT (integrated mutation profiling of actionable cancer targets)
Yin et al[89]	RNA-seq (2 pairs)	RNA-seq based mutation study	Point mutations and indels (Samtools)

¹Description of the software is available at https://confluence.broadinstitute.org/display/CGATools/. NGS: Next-generation sequencing; CRC: Colorectal cancer; TCGA: The cancer genome atlas; MSI: Microsatellite instability.

Table 3 The platforms used in the Cancer Genome Atlas consortium

Alteration types	Glioblastoma multiforme (2008, TCGA)	Colorectal cancers (2012, TCGA)
Point mutations, indels	Sanger sequencing	Illumina GA and HiSeq DNA Sequencing1
		ABI SOLiD DNA Sequencing1
DNA copy numbers	Agilent Human CGH Microarray 244 A	Agilent CGH Microarray Kit 1 × 1 M and 244 A
	Affymetrix Genome-Wide SNP Array 6.0	Affymetrix Genome-Wide SNP Array 6.0
	Illumina Human Infinium 550 K BeadChip	Illumina Infinium 550 K and 1M-Duo BeadChip
DNA Methylation	Illumina Infinium DNA Methylation 27	Illumina Infinium DNA Methylation 27
		Illumina DNA Methylation Cancer Panel I
Transcriptome	Affymetrix Human Genome U133 Plus 2.0	Illumina GA and HiSeq RNA sequencing1
	Agilent 244 K Custom Array	Agilent 244 K Custom Array
	Affymetrix Human Exon 1.0 ST Array
MicroRNA	Agilent 8 × 15 K Human miRNA Microarray	Illumina GA and HiSeq miRNA sequencing1
Whole-genome sequencing	N/A	Illumina HiSeq DNA sequencing1

¹Next-generation sequencing-based platforms are noted. The platforms used in the genomic characterization of glioblastoma multiforme in 2008 (left) and colorectal cancer genomes in 2012 (right) are shown. TCGA: The Cancer Genome Atlas.