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Cheatography

Experimental design Cheat Sheet by

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.
 

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
 

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)

Authors

Rekin’s Janky, rekins.ja­nky­@vib.be (VIB Nucleomics Core, BE)
Judith Boer, j.m.bo­er@­era­smu­smc.nl (Erasmus Medical Centre, NL)
Pete­r-Bram ‘t Hoen, P.A.C._­t_­Hoe­n@l­umc.nl (Leiden University Medical Center, NL)
Wouter Van Delm, wouter.va­nde­lm@­vib.be (VIB Nucleomics Core, BE)

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