Human Whole Exome Sequencing
Kev Pabcuam Nta
● Ob lub exome panels muaj raws li lub hom phiaj enrichment nrog probes: Tseeb Xaiv Tib Neeg Txhua Exon v6 (Agilent) thiab xGen Exome Hybridization Vaj Huam Sib Luag v2 (IDT).
● Sequencing ntawm Illumina NovaSeq.
● Bioinformatic pipeline xa mus rau kev soj ntsuam kab mob lossis kev soj ntsuam qog.
Kev Pabcuam Zoo
●Targets Protein Coding Region: Los ntawm kev ntes thiab ua raws cov cheeb tsam protein coding, hWES yog siv los nthuav tawm cov kev hloov pauv ntsig txog cov qauv protein.
●Nqi Zoo:hWES yields kwv yees li 85% ntawm tib neeg cov kab mob cuam tshuam txog kev hloov pauv los ntawm 1% ntawm tib neeg genome.
●Siab raug: Nrog rau qhov sib sib zog nqus qhov tob, hWES txhawb kev tshawb pom ntawm ob qho tib si sib txawv thiab tsis tshua muaj qhov sib txawv nrog zaus qis dua 1%.
●Rigorous Quality Control: Peb siv tsib lub ntsiab lus tswj hwm thoob plaws txhua theem, los ntawm cov qauv thiab cov tsev qiv ntawv npaj mus rau kev sib txuas lus thiab bioinformatics. Qhov kev soj ntsuam nruj me ntsis ua kom cov khoom xa tuaj raws li cov txiaj ntsig zoo.
●Comprehensive Bioinformatics Analysis: Peb lub raj xa dej mus dhau qhov kev txheeb xyuas qhov hloov pauv mus rau qhov siv genome, vim nws suav nrog kev tshuaj ntsuam xyuas qib siab tsim los tshwj xeeb rau cov lus nug tshawb fawb ntsig txog caj ces ntawm cov kab mob lossis kev tshuaj xyuas qog.
●Kev them nyiaj yug tom qab muag:Peb qhov kev cog lus txuas ntxiv dhau qhov kev ua tiav nrog rau 3 lub hlis tom qab kev muag khoom. Nyob rau lub sijhawm no, peb muab cov phiaj xwm taug qab, kev pab daws teeb meem, thiab Q&A ntu los daws cov lus nug ntsig txog cov txiaj ntsig.
Qauv Specifications
| Exon Capture Strategy | Sequence Strategy | Pom zoo cov ntaub ntawv tso zis |
| Nco ntsoov Xaiv Tib Neeg Txhua Tus Exon v6 (Agilent) los yog xGen Exome Hybridization Vaj Huam Sib Luag v2 (IDT)
| Illumina NovaSeq PE 150 | 5-10 Gb Rau cov kab mob mendelian / cov kab mob tsis tshua muaj:> 50x Rau cov qog nqaij hlav: ≥ 100x |
Cov Qauv Xav Tau
| Hom qauv
| Tus nqi(Qubit®)
| Nco ntsoov | Ntim
| Purity (NanoDrop ™) |
|
Genomic DNA
| ≥ 50 ng | ≥ 6 ng / μL | ≥ 15 μL | OD 260/280 = 1.8-2.0 tsis muaj degradation, tsis muaj paug
|
Bioinformatics
Bioinformatic tsom xam ntawm hWES-kab mob kuaj muaj xws li:
● Sequencing cov ntaub ntawv QC
● Siv Genome Alignment
● Kev txheeb xyuas ntawm SNPs thiab InDels
● Cov Lus Qhia Ua Haujlwm ntawm SNPs thiab InDels
Bioinformatic tsom xam ntawm qog qog muaj xws li:
● Sequencing cov ntaub ntawv QC
● Siv Genome Alignment
● Kev txheeb xyuas ntawm SNPs, InDels thiab somatic variations
● Kev txheeb xyuas cov kab mob germline variants
● Kev txheeb xyuas kev kos npe hloov pauv
● Kev txheeb xyuas cov noob caj noob ces raws li qhov nce ntawm kev ua haujlwm hloov pauv
● Kev hloov pauv cov lus piav qhia ntawm qib ntawm cov tshuaj tsis haum tshuaj
● Heterogeneity tsom xam – xam ntawm purity thiab ploidy
Kev Pabcuam Ua Haujlwm Flow
Qauv xa tuaj
DNA rho tawm
Kev tsim kho tsev qiv ntawv
Sequencing
Cov ntaub ntawv tsom xam
Kev xa cov ntaub ntawv
Kev pab cuam tom qab muag
Cov ntaub ntawv QC - Kev txheeb xyuas ntawm Exome capture
Kev txheeb xyuas qhov sib txawv - InDels
Kev txheeb xyuas qib siab: kev txheeb xyuas thiab kev faib tawm ntawm kev tshem tawm SNPs / InDels - Circos zaj duab xis
Kev tsom xam qog nqaij hlav: kev txheeb xyuas thiab kev faib tawm ntawm kev hloov pauv ntawm somatic - Circos zaj duab xis
Kev tsom xam qog nqaij hlav: clonal kab mob
