NGS CoverageCoverage = Total raw output / Target sequencing size | 50x for de novo sequencing | 30x coverage for resequencing and transcriptomics |
Target sequencing size estimationGENOME | Number of samples x target genome size | AMPLICON | Number of molecules x average molecule size | TRANSCRIPTOME | N genes x average length (bp) |
Genome InformationSpecies | Size | N genes | E. coli | 5 Mbp | 4600 | S. cerevisiae | 13 Mbp | 6000 | A. thaliana | 130 Mbp | 27000 | H. sapiens | 3 Gbp | 25000 |
Variation AnalysisTechnology | WGS | WES | RNA-seq | Mut, Indels | Y | coding | ~coding | CNV | Y | coding | N | Subclonal | N | Y | N | Fusions | ~Y | N | Y | Expression (variants) | N | N | Y |
Filters for somatic variants detection: Paired normal tissue, Minor Allele Frequency (MAF) >0.1% in dbSNP, 1000 genomes, Database of Genomic Variant (DGV).
WES and WGS: report bases not (sufficiently) covered to detect variants. | | INFOMade as support material for the break-out Genomics session - Successful Planning of Large Data Generating Experiments (Leuven, 16 March) |
Sequencing DepthD = Total raw output / read size | Related to coverage, the sequencing depth computes the expected number of reads per sample. | For example, in human : 25,000 genes x 2,000bp x 30 coverage = 1.5Gb per sample. With a single read sequencing of 75 bp reads, this corresponds to 20 M reads per sample. |
Required Sequencing Depth (D)RNA quality | mRNA | total RNA | Good | D | 2D | Degraded | 2D | 4D | Bad | 4D | 8D |
Specifications (Illumina)Platform | Max read length | Max N reads | Output | MiSeq | 2x300 bp | 25 M | 15 Gb | NextSeq Medium | 2x150 bp | 130 M | 39 Gb | NextSeq High | 2x150 bp | 400 M | 120 Gb | HiSeq2500 Rapid | 2x250 bp | 600 M | 300 Gb | HiSeq3000 | 2x150 bp | 2500 M | 750 Gb | HiSeq2500 High | 2x125 bp | 4000 M | 1 Tb | HiSeq4000 | 2x150 bp | 5000 M | 1.5 Tb | HiSeq X Ten | 2x150 bp | 6000 M | 1.8 Tb |
| | Number of samples per applicationPlatform | TotalRNA (40M reads) | WES (>90% >10x) | WGS (30x) | MiSeq | NA | NA | NA | NextSeq Medium | 3 | 3 | NA | NextSeq High | 10 | 10 | 1 | HiSeq2500 Rapid | 15 | 20 | 1 | HiSeq3000 | 50 | 96 | 6 | HiSeq2500 High | 100 | 150 | 10 | HiSeq4000 | 125 | 180 | 12 | HiSeq X Ten | NA | NA | 16 |
Costs per sample (Library prep + NGS)Cost | Application | ~€4000 | WGS 30x tumor, 10x germline (2x125 bp) | ~€2500 | WES 200x tumor, 100x germline (2x125 bp) | ~€750 | Total RNA seq, 20-30 M reads, stranded library, rRNA depleted (2x125 bp) | ~€300 | mRNA seq, 15-20 M reads, stranded library (75 bp) |
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