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T2T Genome Sib Dhos | Ultra Long Sequencing
T2T (Telomere-rau-Telomere) Genome yog tus qauv kub rau kev sib sau ua ke genome zoo, hais txog kev tsim kho genome uas tsis muaj qhov sib txawv lossis tsis muaj qhov sib txawv, uas nthuav dav ntawm ib qho telomere mus rau lwm qhov, thiab rhuav tshem cov kev txwv ntawm kev sib sau ua ke genome ib txwm muaj.
Siv lub zog los ntawm core ONT ultra-long read sequencing thiab kev sib xyaw nrog ntau lub platform deep sequencing thiab cov optimized bioinformatics pipelines, BMKGENE T2T Genome kev daws teeb meem tsom mus rau cov genomic "tsaus ntuj" uas nyuaj tshaj plaws - telomeres (cov nucleoprotein complexes tshwj xeeb ntawm eukaryotic chromosome kawg), cov kab mob siab dua centromeres (cov tandem repeat arrays loj heev), thiab lwm cov complex repeat thiab heterozygous haplotype regions, uas tau ntev tsis daws tau rau cov standard long-read sequencing. Tsis zoo li cov standard long reads uas tsis hla cov regions no thiab ua rau cov sequencing collapse lossis chimeric contigs, ONT ultra-long reads tuaj yeem hla cov unassembleable gaps thiab complex regions. BMKGene cog lus tias yuav xa cov gap-free lossis gap-less, high-quality T2T genomes rau ntau hom tsiaj.
Kev tsim cov T2T genome qhib cov cheeb tsam genomic nyuaj uas yav dhau los tsis tau mus txog, ua kom tiav cov kev tshawb fawb tseem ceeb, thiab muab cov ntaub ntawv ruaj khov, siab raug rau kev tshawb fawb tob tob suav nrog kev hloov pauv ntawm cov tsiaj txhu, kev tshawb nrhiav cov noob caj noob ces, kev yug me nyuam molecular, kev kho mob kom raug thiab lwm yam kev tshawb fawb tshiab.
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Kev kawm txog Proteomics
Proteomics tsom mus rau cov protein—cov neeg ua haujlwm ntawm lub neej ua ub ua no uas ua lub luag haujlwm tseem ceeb hauv kev tswj hwm kev sau ntawv ntawm lub cev. Nws tshuaj xyuas cov khoom sib xyaw, cov qib kev qhia tawm, thiab cov xwm txheej hloov pauv ntawm txhua cov protein hloov pauv hauv cov ntaub so ntswg lossis cov hlwb, hais txog qhov cuam tshuam tseem ceeb ntawm cov protein ntau ntawm cov txheej txheem hauv lub neej. Siv dav hauv kev kho mob, kev ua liaj ua teb, thiab kev yug tsiaj. Cov Proteomics zoo siv cov thev naus laus zis txheeb xyuas cov protein HPLC-MS/MS los txheeb xyuas cov qauv suav nrog cov gel strips, IP, thiab CO-IP/Pull down samples. Quantitative Proteomics ua tiav qhov tseeb ntawm kev ntsuas thiab kev txheeb xyuas ntawm txhua cov protein uas qhia los ntawm genome lossis hauv ib qho system sib xyaw ua ke. Cov thev naus laus zis proteomics tam sim no feem ntau yog muab faib ua cov cim (TMT) thiab cov cim tsis muaj (Label Free, DIA, PRM). BMKGENE muab ntau lub platform thiab ntau cov thev naus laus zis proteomics kev daws teeb meem.
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Kev Tshawb Fawb Txog Metabolomics
Kev kawm txog kev hloov pauv ntawm cov metabolism, uas yog ib qho kev kawm txuas ntxiv ntawm genomics, feem ntau yog tsom mus rau cov tshuaj me me uas muaj qhov hnyav molecular tsawg dua 1500 Da. Nws ua rau cov metabolites muaj peev xwm cuam tshuam cov kab mob cov lus teb rau cov kev hloov pauv sab nraud thiab kev hloov pauv ntawm lub cev/kev mob nkeeg tau zoo dua. Kev hloov pauv ntawm cov metabolism uas ua rau muaj kev hloov pauv ntawm cov noob caj noob ces kuj yog ib qho kev tshawb fawb tshiab, muab ib qho kev xav tshiab rau kev tshawb fawb.
BMKGENE muab ntau yam kev pabcuam metabolomics, suav nrog cov metabolomics uas tsis yog lub hom phiaj, cov metabolomics uas tau tsom mus rau ntau yam, thiab cov metabolomics uas tau tsom mus rau lub hom phiaj. Siv cov kua chromatography-mass spectrometry (LC-MS) lossis roj chromatography-mass spectrometry (GC-MS), cov kev hloov pauv dynamic hauv feem ntau cov metabolites me me hauv cov kab mob ua ntej thiab tom qab kev txhawb nqa sab nraud tuaj yeem pom. Lub hauv paus ntawm cov kev pabcuam no yog nyob rau hauv kev txheeb xyuas cov metabolites nrog qhov sib txawv tseem ceeb ntawm cov pab pawg sim thiab cov pab pawg tswj thiab tshawb nrhiav ntxiv lawv txoj kev sib raug zoo nrog kev hloov pauv physiological / pathological thiab cov txheej txheem hauv qab.
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Exosomal mRNA/LncRNA/CircRNA Sequencing-Illumina
Exosomes yog cov vesicles me me uas cov cell tso tawm, feem ntau muaj qhov loj li ntawm 30 txog 100 nanometers. Cov vesicles no muaj ntau yam RNA. Exosomes ntseeg tau tias ua lub luag haujlwm tseem ceeb hauv kev sib txuas lus ntawm cov cell, kev tiv thaiv kab mob, thiab kev loj hlob ntawm kab mob, thiab tuaj yeem kis mus rau lwm qhov ntawm lub cev los ntawm cov kua dej hauv lub cev xws li plasma, qaub ncaug, thiab zis. Lawv nqa cov biomolecules tshwj xeeb los tswj cov haujlwm ntawm cov cell txais, cuam tshuam rau cov xwm txheej cellular physiological. Exosomes kuj raug suav hais tias ua lub luag haujlwm tseem ceeb hauv kev loj hlob ntawm kab mob, suav nrog mob qog noj ntshav, kab mob neurodegenerative, thiab cov mob inflammatory. Kev tshawb fawb txog exosomes muab cov kev nkag siab tshiab thiab cov txheej txheem rau kev kuaj mob, kev kho mob, thiab kev tiv thaiv kab mob.
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Kev Tshawb Fawb Txog RNA Me Me Exosomal-Illumina
Exosomes yog cov vesicles me me uas cov cell tso tawm, feem ntau muaj txoj kab uas hla ntawm 30 txog 100 nanometers. Cov vesicles no muaj ntau yam RNA. Ntawm cov hom RNA hauv exosomes, qhov feem ntau thiab kawm ntau yog microRNA (miRNA). miRNA yog ib chav kawm ntawm cov RNAs me me uas tsis yog coding thiab kwv yees li 18-25 nucleotides ntev. Lawv ua haujlwm nruab nrab ntawm kev kaw cov noob caj noob ces tom qab kev sau los ntawm kev khi rau 3′ untranslated cheeb tsam (3′ UTR) ntawm cov mRNAs uas xav tau, yog li tswj kev qhia noob caj noob ces. Piv txwv li, exosomes uas qee cov cell qog nqaij hlav tso tawm muaj cov miRNAs tshwj xeeb, xws li miR-126 thiab miR-92a. Cov miRNAs no tuaj yeem cuam tshuam kev qhia noob caj noob ces hauv cov cell txais thiab txhawb kev loj hlob ntawm qog nqaij hlav (Tomohiro Umezu, et al., Oncogene, 2012).
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BMKMANU S3000_Spatial Transcriptome
Kev sau ntawv txog qhov chaw nyob yog ib txoj kev siv uas cia peb ntes thiab pom cov noob caj noob ces hauv cov nqaij. Qhov no tseem ceeb heev rau kev nkag siab txog kev sib cuam tshuam ntawm cov hlwb.
Muaj ntau lub platform sib txawv rau txoj hauv kev no. Hauv qhov teeb meem no, BMKGene tau tsim BMKManu 3000 Spatial transcriptome Chip, lub platform uas txhawb nqa kev ua haujlwm ntawm cov txheej txheem, ncav cuag kev daws teeb meem subcellular thiab ua kom muaj ntau theem kev daws teeb meem.
Cov chip no muaj 4.2 lab qhov chaw siv cov thev naus laus zis patented ntawm microwells txheej nrog cov hlaws dai uas muaj cov probes barcoded spatially. Nrog txoj kev no, tom qab ntes thiab amplification, peb tau txais lub tsev qiv ntawv cDNA nplua nuj nrog cov qauv barcoded uas nws yog Illumina sib xws.
Ntawm cov ntaub ntawv, kev sib xyaw ua ke ntawm qhov chaw barcode thiab UMIs ua kom ntseeg tau qhov tseeb thiab qhov tshwj xeeb ntawm cov ntaub ntawv tsim tawm. Kev sib xyaw ua ke tag nrho cov saum toj no, BMKManu muab cov ntaub ntawv teeb tsa ntau yam.
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Cov tsev qiv ntawv DNBSEQ uas tau ua tiav lawm
DNBSEQ, tsim los ntawm MGI, yog ib qho thev naus laus zis NGS tshiab uas tau tswj kom txo qis tus nqi sequencing thiab ua kom muaj txiaj ntsig ntau dua. Kev npaj cov tsev qiv ntawv DNBSEQ suav nrog DNA fragmentation, kev npaj ssDNA, thiab dov lub voj voog amplification kom tau txais DNA nanoballs (DNB). Cov no tom qab ntawd raug thauj mus rau ntawm qhov chaw khov kho thiab tom qab ntawd sequenced los ntawm combinatorial Probe-Anchor Synthesis (cPAS). DNBSEQ thev naus laus zis sib xyaw ua ke cov txiaj ntsig ntawm kev muaj qhov yuam kev amplification qis nrog kev siv cov qauv yuam kev ceev ceev nrog nanoballs, ua rau sequencing nrog throughput siab dua thiab raug.
Peb qhov kev pabcuam sequencing uas tau ua tiav lawm ua rau cov neeg siv khoom tuaj yeem npaj Illumina sequencing libraries los ntawm ntau qhov chaw sib txawv (mRNA, tag nrho genome, amplicon, 10x libraries, thiab lwm yam), uas tau hloov mus rau MGI libraries hauv peb lub chaw soj nstuam kom raug sequenced hauv DNBSEQ-T7, ua rau muaj cov ntaub ntawv ntau ntawm cov nqi qis dua.
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Kev Sib Cuam Tshuam ntawm Chromatin raws li Hi-C
Hi-C yog ib txoj kev tsim los ntes cov qauv genomic los ntawm kev sib txuas ua ke probing proximity-based interactions thiab high-throughput sequencing. Txoj kev no yog raws li chromatin crosslinking nrog formaldehyde, ua raws li kev zom thiab rov ua dua tshiab hauv txoj kev uas tsuas yog cov khoom uas tau txuas nrog covalently thiaj li yuav tsim cov khoom ligation. Los ntawm kev sequencing cov khoom ligation no, nws muaj peev xwm kawm txog 3D lub koom haum ntawm genome. Hi-C ua rau kev kawm txog kev faib tawm ntawm cov feem ntawm genome uas tau ntim me me (A compartments, euchromatin) thiab yuav muaj kev ua haujlwm transcriptional, thiab cov cheeb tsam uas tau ntim nruj dua (B compartments, Heterochromatin). Hi-C kuj tseem siv tau los taw qhia Topologically Associated Domains (TADs), cov cheeb tsam ntawm genome uas muaj cov qauv tais thiab yuav muaj cov qauv qhia zoo sib xws, thiab los txheeb xyuas cov chromatin loops, DNA cheeb tsam uas tau anchored ua ke los ntawm cov protein thiab uas feem ntau muaj ntau yam hauv cov ntsiab lus tswj hwm. BMKGene qhov kev pabcuam Hi-C sequencing ua rau cov kws tshawb fawb muaj peev xwm tshawb nrhiav qhov chaw ntawm genomics, qhib txoj hauv kev tshiab rau kev nkag siab txog kev tswj hwm genome thiab nws cov txiaj ntsig rau kev noj qab haus huv thiab kab mob.
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PacBio 2 + 3 Kev daws teeb meem mRNA puv ntoob
Txawm hais tias NGS-based mRNA sequencing yog ib qho cuab yeej siv tau ntau yam rau kev ntsuas qhov kev qhia tawm ntawm cov noob caj noob ces, nws txoj kev vam khom rau kev nyeem luv luv txwv nws txoj kev ua haujlwm hauv kev tshuaj xyuas cov ntawv sau ua ke. Ntawm qhov tod tes, PacBio sequencing (Iso-Seq) siv cov thev naus laus zis nyeem ntev, ua rau muaj kev txheeb xyuas cov ntawv sau mRNA tag nrho. Txoj hauv kev no pab txhawb kev tshawb nrhiav txog lwm txoj hauv kev splicing, gene fusions, thiab poly-adenylation, txawm hais tias nws tsis yog qhov kev xaiv tseem ceeb rau kev ntsuas qhov kev qhia tawm ntawm cov noob caj noob ces. Kev sib xyaw ua ke 2 + 3 txuas qhov sib txawv ntawm Illumina thiab PacBio los ntawm kev vam khom PacBio HiFi nyeem kom paub txog cov txheej txheem tiav ntawm cov ntawv sau isoforms thiab NGS sequencing los ntsuas cov isoforms zoo sib xws.
Platforms: PacBio Revio thiab Illumina NovaSeq
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Kev Tshawb Fawb Txog Kev Koom Tes Thoob Plaws Genome
Lub hom phiaj ntawm Genome-Wide Association Studies (GWAS) yog txhawm rau txheeb xyuas cov kev hloov pauv caj ces (genotypes) txuas nrog cov cwj pwm tshwj xeeb (phenotypes). Los ntawm kev tshuaj xyuas cov cim caj ces thoob plaws tag nrho cov genome hauv ntau tus neeg, GWAS nthuav dav genotype-phenotype kev sib raug zoo los ntawm kev tshuaj xyuas cov pej xeem. Txoj kev no pom cov ntawv thov dav dav hauv kev tshawb fawb txog cov kab mob tib neeg thiab tshawb nrhiav cov noob caj ces ua haujlwm ntsig txog cov cwj pwm nyuaj hauv tsiaj lossis nroj tsuag.
Ntawm BMKGENE, peb muaj ob txoj hauv kev rau kev ua GWAS ntawm cov pej xeem coob: siv Whole-Genome Sequencing (WGS) lossis xaiv txoj kev txo qis kev sawv cev genome sequencing, uas yog tsim los ntawm tsev Specific-Locus Amplified Fragment (SLAF). Txawm hais tias WGS haum rau cov genomes me dua, SLAF tshwm sim ua lwm txoj hauv kev pheej yig rau kev kawm cov pej xeem coob dua nrog cov genomes ntev dua, txo cov nqi sequencing, thaum lav qhov kev tshawb pom cov cim caj ces siab.
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Kev Txheeb Xyuas RNA Ib Leeg
Kev tsim cov txheej txheem ntawm kev ntes ib lub cell thiab kev tsim cov tsev qiv ntawv, ua ke nrog kev ua kom muaj txiaj ntsig zoo, tau hloov pauv txoj kev tshawb fawb txog kev qhia txog noob caj noob ces ntawm theem cell. Qhov kev tshawb pom no tso cai rau kev tshuaj xyuas tob dua thiab ntau dua ntawm cov pej xeem cell nyuaj, kov yeej cov kev txwv uas cuam tshuam nrog kev nruab nrab ntawm kev qhia txog noob caj noob ces hla txhua lub cell thiab khaws cia qhov sib txawv tiag tiag hauv cov pej xeem no. Thaum kev ua kom muaj noob caj noob ces ib lub cell (scRNA-seq) muaj qhov zoo tsis lees paub, nws ntsib cov teeb meem hauv qee cov nqaij uas kev tsim cov noob caj noob ces ib lub cell nyuaj thiab xav tau cov qauv tshiab. Ntawm BMKGene, peb daws qhov teeb meem no los ntawm kev muab cov noob caj noob ces ib lub nucleus RNA (snRNA-seq) siv cov thev naus laus zis 10X Genomics Chromium uas siv niaj hnub no. Txoj hauv kev no ua rau cov qauv ntawm cov qauv dav dua rau kev tshuaj xyuas transcriptome ntawm theem cell ib lub cell.
Kev rho tawm cov nuclei tau ua tiav los ntawm lub 10X Genomics Chromium chip tshiab, uas muaj yim-channel microfluidics system nrog ob chav hla. Hauv lub system no, cov hlaws gel uas muaj cov barcodes, primers, enzymes, thiab ib lub nucleus raug encapsulated hauv nanoliter-sized roj tee, tsim Gel Bead-in-Emulsion (GEM). Tom qab GEM tsim, cell lysis thiab barcode tso tawm tshwm sim hauv txhua GEM. Tom qab ntawd, mRNA molecules dhau los ua reverse transcription rau hauv cDNAs, suav nrog 10X barcodes thiab Unique Molecular Identifiers (UMIs). Cov cDNAs no tom qab ntawd raug rau kev tsim cov tsev qiv ntawv sequencing txheem, pab txhawb kev tshawb nrhiav zoo thiab kev tshawb nrhiav ntawm cov qauv qhia txog gene ntawm ib lub cell.
Platform: 10 × Genomics Chromium thiab Illumina NovaSeq Platform
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Kev Txheeb Xyuas Tag Nrho Cov Genome ntawm Cov Nroj Tsuag/Tsiaj
Whole Genome Sequencing (WGS) yog ib txoj kev siv los txiav txim siab tag nrho cov DNA sib lawv liag ntawm ib yam khoom hauv ib lub sijhawm.
Feem ntau, cov kev pabcuam raug muab faib ua ob pawg sib txawv nyob ntawm seb muaj cov genome siv li cas:
- De novokev txheeb xyuas tag nrho cov genome.Hauv qhov xwm txheej no, lub genome uas yuav tsum tau sequenced tsis muaj ib qho genome siv tau, thiab vim li ntawd, lub hom phiaj ntawm qhov sequencing no yog los tsim nws (lossis txhim kho ib qho uas twb muaj lawm). Cov txheej txheem no yuav tsum siv ob qho tib si, Illumina cov ntaub ntawv thiab kev nyeem ntev sequencing los txhim kho cov genome sib dhos los ntawm kev tsim kom muaj kev sib tshooj ntawm kev nyeem.
- Kev rov ua dua qhov kev txheeb xyuas.Nws yog hais txog tag nrho cov genome sequencing ntawm cov tib neeg sib txawv ntawm cov tsiaj txhu uas paub cov genomes siv. Los ntawm qhov no, qhov sib txawv ntawm genomic ntawm cov tib neeg lossis cov pej xeem tuaj yeem txheeb xyuas ntxiv.
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Kev Tshawb Fawb Txog MRNA Tag Nrho-Nanopore
Txawm hais tias NGS-based mRNA sequencing yog ib qho cuab yeej siv tau ntau yam rau kev ntsuas qhov kev qhia tawm ntawm cov noob caj noob ces, nws txoj kev vam khom rau kev nyeem luv luv txwv nws txoj kev ua haujlwm hauv kev tshuaj xyuas cov ntawv sau ua ke. Ntawm qhov tod tes, nanopore sequencing siv cov thev naus laus zis nyeem ntev, ua rau muaj kev txheeb xyuas cov ntawv sau mRNA tag nrho. Txoj hauv kev no pab txhawb kev tshawb nrhiav txog lwm txoj hauv kev splicing, gene fusions, poly-adenylation, thiab kev ntsuas ntawm mRNA isoforms.
Kev ntsuas Nanopore sequencing, ib txoj kev uas siv cov teeb liab hluav taws xob nanopore ib lub molecule tiag tiag, muab cov txiaj ntsig tiag tiag. Kev coj los ntawm cov protein motor, ob txoj hlua DNA khi rau cov protein nanopore uas muab tso rau hauv biofilm, unwinding thaum nws hla dhau lub nanopore channel nyob rau hauv qhov sib txawv voltage. Cov teeb liab hluav taws xob sib txawv uas tsim los ntawm cov hauv paus sib txawv ntawm DNA strand raug kuaj pom thiab faib ua pawg tiag tiag, pab txhawb kev ntsuas nucleotide kom raug thiab txuas ntxiv mus. Txoj hauv kev tshiab no kov yeej cov kev txwv luv luv thiab muab lub platform dynamic rau kev tshuaj xyuas genomic nyuaj, suav nrog kev tshawb fawb transcriptomic nyuaj, nrog cov txiaj ntsig tam sim ntawd.
Platform: Nanopore PromethION 48
