2. カバー率 = (N × L) / G : 1塩基当りの断片数
リード数 : N
リード数 : L
ゲノム長 : G
…AGGTGCATGCCGCATCGATCGAGC…
AGGTGCATG
GCATGCCGCAT
GCATCGATCGAGC
paired end
single end
ゲノム
リード
Next Generation Sequencer (NGS)
DNAを断片化して配列を読む→参照ゲノムにマッピング→配列を再構成する
カバー率が十分でないと正確な配列情報が得られない。
3. ? Exon : 1~2% of the genome
? SureSelectXT Human All Exon Kit V6 (Agilent)
Whole-Exome Sequencing (WES) 解析
Agilent ?
7. ? VarScan2
VarScan 2: Somatic mutation and copy number alteration
discovery in cancer by exome sequencing.
Koboldt, D., et al. Genome Res. 2012 22:568-576
http://varscan.sourceforge.net/
Estimation of the Somatic Mutations
8. A strategy for estimation of the somatic mutations
VarScan 2: Somatic mutation and copy number alteration discovery in cancer by exome
sequencing.
Koboldt, D., et al.Genome Res. 2012 22:568-576
Normal vs Non-micropapillary
Normal vs MPA
12. Variant Call Format (VCF)
#CHROM POS ID REF ALT QUAL FILTER
chr1 14930 2 A G 0.042602 PASS
chr1 1455924 3 T C 0.067266 PASS
chr1 2059684 4 G A 0.05858 PASS
chr1 6554475 5 G A 0.035576 PASS
chr1 10413139 6 C T 0.055485 PASS
chr1 11668837 7 G A 0.024802 PASS
chr1 16058680 8 G A 0.067123 PASS
chr1 16563946 9 C T 0.04199 PASS
#8: The somatic mutations are estimated using varscan2 released from Koboldt et al.
#9: This illustration indicates the estimation-strategy of varscan2. Single nucleotide variations are compared between normal and tumor cell. When probability of mutation in tumor cell is significantly higher than that in normal cell, it is estimated as the somatic mutation. The somatic mutations in non maicropapillary and micropapillary tumor are estimated by using varscan2.
