NGS Coverage
Coverage = Total raw output / Target sequencing size |
50x for de novo sequencing |
30x coverage for resequencing and transcriptomics |
Target sequencing size estimation
GENOME |
Number of samples x target genome size |
AMPLICON |
Number of molecules x average molecule size |
TRANSCRIPTOME |
N genes x average length (bp) |
Genome Information
Species |
Size |
N genes |
E. coli |
5 Mbp |
4600 |
S. cerevisiae |
13 Mbp |
6000 |
A. thaliana |
130 Mbp |
27000 |
H. sapiens |
3 Gbp |
25000 |
Variation Analysis
Technology |
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.
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INFO
Made as support material for the break-out Genomics session - Successful Planning of Large Data Generating Experiments (Leuven, 16 March) |
Sequencing Depth
D = 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 |
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Number of samples per application
Platform |
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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