Tsarin Shuka/ Dabba De Novo Genome Sequencing
De NovoJerin abubuwa yana nufin gina dukkan kwayoyin halittar wani nau'in ta amfani da fasahar jeri ba tare da wani kwayar halitta ba. Gabatarwa da kuma amfani da tsarin jeri na ƙarni na uku, wanda ke ɗauke da tsawon karatu, ya inganta haɗuwar kwayoyin halitta sosai ta hanyar ƙara haɗuwa tsakanin karatu. Wannan haɓakawa yana da mahimmanci musamman lokacin da ake mu'amala da kwayoyin halitta masu ƙalubale, kamar waɗanda ke nuna babban heterozygosity, babban rabo na yankuna masu maimaitawa, polyploids, da yankuna tare da abubuwan da ke maimaitawa, abubuwan da ke cikin GC marasa kyau, ko babban rikitarwa waɗanda yawanci ba a haɗa su da kyau ta amfani da tsarin jeri na ɗan gajeren lokaci kawai.
Maganinmu na tsayawa ɗaya yana ba da ayyukan haɗa jerin abubuwa da nazarin bioinformatic wanda ke samar da ingantaccen kwayar halittar de novo da aka haɗa. Wani bincike na farko game da kwayar halittar Illumina yana ba da kimanta girman kwayar halittar da sarkakiya, kuma ana amfani da wannan bayanin don jagorantar mataki na gaba na jerin abubuwan da aka karanta na dogon lokaci tare da PacBio HiFi, sannan kumana sabuwar shekarahaɗuwar ƙwayoyin cuta. Amfani da tarin ƙwayoyin cuta na HiC daga baya yana ba da damar haɗa ƙwayoyin cuta zuwa ga kwayar halitta, ta hanyar samun haɗuwar matakin chromosome. A ƙarshe, ana yin bayanin kwayar halitta ta hanyar hasashen kwayoyin halitta da kuma jerin kwayoyin halitta da aka bayyana, ta hanyar amfani da transcriptomes masu gajeru da dogon karatu.
Fasallolin Sabis
● Haɗa ayyukan jerin ayyuka da yawa da kuma ayyukan bioinformatic a cikin mafita ɗaya-ɗaya:
Binciken kwayoyin halitta tare da Illumina don kimanta girman kwayoyin halitta da kuma jagorantar matakai na gaba;
Tsarin karatu mai tsawo donna sabuwar shekarataron ƙwayoyin cuta;
Tsarin Hi-C don ɗaure ƙwayoyin chromosome;
jerin mRNA don bayanin kwayoyin halitta;
Tabbatar da taron.
● Sabis ɗin da ya dace da gina sabbin kwayoyin halitta ko inganta kwayoyin halitta masu alaƙa da ake da su don nau'ikan halittu masu ban sha'awa.
Fa'idodin Sabis
Ci gaban dandamalin jerin abubuwa da kuma ilimin halittu a cikinna sabuwar shekarataron kwayoyin halitta
(Amarasinghe SL da sauransu,Ilimin Halittar Kwayoyin Halitta, 2020)
●Kwarewa Mai Zurfi da Bayanan BugawaBMKGene ta tara kwarewa mai yawa a cikin tarin kwayoyin halitta masu inganci na nau'ikan halittu daban-daban, gami da kwayoyin halittar diploid da kwayoyin halittar da ke da rikitarwa na nau'ikan polyploid da allopolyploid. Tun daga shekarar 2018, mun ba da gudummawa ga fiye daLittattafai 300 masu tasiri sosai, kuma sama da 20 daga cikinsu an buga su a cikin Nature Genetics.
● Mafita Mai Tsaya Ɗaya: tsarinmu na haɗaka ya haɗa fasahohin tsara jerin abubuwa da nazarin halittu cikin tsarin aiki mai haɗin kai, yana samar da ingantaccen tsarin halittar da aka haɗa.
●An daidaita shi da buƙatunku: Tsarin aikinmu na hidima yana da sauƙin daidaitawa, yana ba da damar daidaitawa ga kwayoyin halitta masu siffofi daban-daban da takamaiman buƙatun bincike. Wannan ya haɗa da ɗaukar manyan kwayoyin halitta, kwayoyin halitta na polyploid, kwayoyin halitta masu heterozygous sosai, da ƙari.
●Ƙwararrun ...tare da kyakkyawar gogewa a fannin gwaji da kuma bioinformatics na hadaddun kwayoyin halitta da kuma jerin haƙƙin mallaka da software.
●Tallafin Bayan Talla:Alƙawarinmu ya wuce kammala aikin da tsawon watanni 3 bayan an sayar da shi. A wannan lokacin, muna ba da shawarwari kan yadda za a bi diddigin aikin, taimakawa wajen magance matsaloli, da kuma zaman tambayoyi da amsoshi don magance duk wata tambaya da ta shafi sakamakon.
Bayanin Sabis
| Binciken kwayoyin halitta | Haɗakar kwayoyin halitta | Matakan Chromosome | Bayanin Halittar Jini |
| 50X Illumina NovaSeq PE150
| 30X PacBio CCS HiFi yana karantawa | 100X Hi-C | RNA-seq Illumina PE150 10 Gb + (zaɓi ne) Cikakken tsayin RNA-seq PacBio 40 Gb ko Nanopore 12 Gb |
Bukatun Sabis
Don Binciken Kwayoyin Halitta, Taro na Kwayoyin Halitta da Taro na Hi-C:
| Nama ko kuma nucleic acid da aka cire | Binciken Kwayoyin Halitta | Haɗakar Genome tare da PacBio | Babban Taro na Hi-C |
| Viscera na Dabbobi | 0.5-1 g
| ≥ 3.5 g | ≥2 g |
| Tsokar Dabbobi | ≥ 5 g | ||
| Jinin Dabbobi Masu Shayarwa | 1.5 mL
| ≥ 5 mL | ≥2 mL |
| Jinin Kaji/Kifi | ≥ 0.5 mL | ||
| Shuke-Sabon Ganyen Ganyayyaki | 1-2 g | ≥ 5 g | ≥ 4 g |
| Kwayoyin Halitta Masu Al'adu |
| ≥ 1x108 | ≥ 1x107 |
| Kwari | 0.5-1 g | ≥ 3 g | ≥ 2 g |
| DNA da aka cire | Mayar da hankali: ≥1 ng/µL Adadin ≥ 30 ng Iyakance ko babu lalacewa ko gurɓatawa | Mayar da hankali: ≥ 50 ng/ µL Adadi: 10 µg/ƙwayar kwarara/samfuri OD260/280=1.7-2.2 OD260/230=1.8-2.5 Iyakance ko babu lalacewa ko gurɓatawa |
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Don ƙarin bayani game da kwayoyin halitta tare da transcriptomics:
| Nama ko kuma nucleic acid da aka cire | Rubutun Illumina | Rubutun PacBio | Rubutun Nanopore |
| Shuke- Tushen/Tushen/Feti | 450 MG | 600 MG | |
| Shuka - Ganye/Iri | 300 MG | 300 MG | |
| Shuka - 'Ya'yan itace | 1.2 g | 1.2 g | |
| Zuciyar Dabbobi/Hanta | 300 MG | 300 MG | |
| Tashin Jiki/Kwakwalwa na Dabbobi | 240 MG | 240 MG | |
| Tsokar Dabbobi | 450 MG | 450 MG | |
| Kasusuwan Dabbobi/Gashi/Fata | 1 g | 1 g | |
| Arthropod - Kwari | 6 | 6 | |
| Arthropod - Crustacea | 300 MG | 300 MG | |
| Jini gaba ɗaya | Bututu 1 | Bututu 1 | |
| RNA da aka cire | Mayar da hankali: ≥ 20 ng/ µL Adadin ≥ 0.3 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥ 6 5≥28S/18S≥1 | Matsakaicin hankali: ≥ 100 ng/µL Adadin ≥ 0.75 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥ 8 5≥28S/18S≥1 | Matsakaicin hankali: ≥ 100 ng/µL Adadin ≥ 0.75 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥ 7.5 5≥28S/18S≥1 |
Isarwa Samfurin da Aka Ba da Shawara
Akwati: Bututun centrifuge na 2 ml (Ba a ba da shawarar a saka tin foil ba)
(Ga yawancin samfuran, muna ba da shawarar kada a adana a cikin ethanol.)
Lakabi da Samfura: Dole ne a yi wa samfuran lakabi a sarari kuma su yi daidai da fom ɗin bayanin samfurin da aka gabatar.
Jigilar Kaya: Busasshen Kankara: Da farko ana buƙatar a saka samfuran a cikin jakunkuna sannan a binne su a cikin busasshen kankara.
Tsarin aiki
Tsarin Aikin Sabis
Tsarin gwaji
Isarwa samfurin
Cire DNA
Gina ɗakin karatu
Jerin abubuwa
Nazarin bayanai
Ayyukan bayan-sayarwa
Cikakken nazarin bioinformatic, an raba shi cikin matakai 4:
1) Binciken kwayoyin halitta, wanda ya dogara da nazarin k-mer tare da NGS, ya karanta:
Kimanta girman kwayar halitta
Kimantawar heterozygosity
Kimanta yankuna masu maimaitawa
2) Haɗakar Genome tare da PacBio HiFi:
Daga sabon zamanitaro
Kimantawar Haɗawa: gami da nazarin BUSCO don cikar kwayar halitta da taswirar bayan NGS da karatun PacBio HiFi
3) Haɗa Hi-C:
QC na ɗakin karatu na Hi-C: kimanta hulɗar Hi-C masu inganci
Haɗawar Hi-C: tattara ƙwayoyin cuta a cikin ƙungiyoyi, sannan a bi tsarin contig a cikin kowace ƙungiya da kuma sanya alkiblar contig
Kimantawa Mai Kyau-C
4) Bayanin kwayoyin halitta:
Hasashen RNA mara coding
Gano jerin maimaitawa (transposons da maimaitawa tandem)
Hasashen kwayar halitta
§Daga sabon zamani: ab initio algorithms
§ Dangane da daidaito
§ An yi shi ne bisa ga rubuce-rubuce, tare da dogaye da gajerun karatu: karatu sunana sabuwar shekaraan haɗa ko an tsara shi zuwa ga daftarin tsarin halittar
§ Bayanin kwayoyin halittar da aka annabta tare da bayanai da yawa
1) Binciken Halittar Jini - nazarin k-mer
2) Tarin Kwayoyin Halitta
2) Haɗakar Genome - PacBio HiFi yana karanta taswira zuwa ga daftarin taron
2) Haɗakar Hi-C - kimantawa na haɗin gwiwar Hi-C masu inganci
3) Kimantawa bayan haɗawa da Hi-C
4) Bayanin Halittar Jini - haɗakar kwayoyin halittar da aka annabta
4) Bayanin kwayoyin halitta - bayanin kwayoyin halitta da aka annabta
Bincika ci gaban da ayyukan haɗa kwayoyin halitta na BMKGene de novo suka samar ta hanyar tarin wallafe-wallafe da aka tsara:
Li, C. da sauransu (2021) 'Jerin kwayoyin halitta suna bayyana hanyoyin warwatsewa na duniya kuma suna ba da shawarar daidaitawar kwayoyin halitta masu haɗuwa a cikin juyin halittar dokin teku', Nature Communications, 12(1). doi: 10.1038/S41467-021-21379-X.
Li, Y. da sauransu (2023) 'Sauye-sauyen chromosomal masu girma-girma suna haifar da canje-canje a yanayin bayyanar kwayoyin halitta, daidaitawar muhalli, da kuma bambancin halittu a cikin Gayal (Bos frontalis)', Ilimin Halittar Kwayoyin Halitta da Juyin Halitta, 40(1). doi: 10.1093/MOLBEV/MSAD006.
Tian, T. da sauransu (2023) 'Tarin kwayoyin halitta da rarraba kwayoyin halitta na wani sanannen ƙwayar masara mai jure fari', Nature Genetics 2023 55:3, 55(3), shafi na 496–506. doi: 10.1038/s41588-023-01297-y.
Zhang, F. et al. (2023) 'Bayyana juyin halittar alkaloid na tropane ta hanyar nazarin kwayoyin halitta guda biyu a cikin dangin Solanaceae', Nature Communications 2023 14:1, 14(1), shafi na 1–18. doi: 10.1038/s41467-023-37133-4.
Nazarce-nazarcen shari'o'i masu ƙalubale:
Haɗa Telomere-zuwa-telomere:Fu, A. da sauransu (2023) 'Tarin kwayoyin halittar Telomere-zuwa-telomere na kankana mai ɗaci (Momordica charantia L. var. abbreviata Ser.) ya bayyana ci gaban 'ya'yan itace, abun da ke ciki da kuma halayen kwayoyin halitta masu nuna', Binciken Noma, 10(1). doi: 10.1093/HR/UHAC228.
Haɗa Haplotype:Hu, W. da sauransu (2021) 'Generum da aka ayyana ta hanyar allele yana bayyana bambancin biallelic a lokacin juyin halittar rogo', Molecular Plant, 14(6), shafi na 851–854. doi: 10.1016/j.molp.2021.04.009.
Babban tarin kwayoyin halitta:Yuan, J. da sauransu (2022) 'Tushen kwayoyin halitta na giga-chromosomes da giga-genome na itacen peony Paeonia ostii', Nature Communications 2022 13:1, 13(1), shafi na 1–16. doi: 10.1038/s41467-022-35063-1.
Haɗakar kwayoyin halittar polyploid:Zhang, Q. da sauransu (2022) 'Bayanin kwayoyin halitta game da raguwar ƙwayoyin halitta na autopolyploid sugarcane Saccharum spontaneum' kwanan nan, Nature Genetics 2022 54:6, 54(6), shafi na 885–896. doi: 10.1038/s41588-022-01084-1.
