DNA/RNA sequencing - PacBio Sequencer
Nta
Ob hom kev sib txuas ntawm PacBio sequencer: Nruam Nyeem Ntev (CLR) thiab Kev Pom Zoo Nyeem Ntawv (CCS)
| Sequence hom | Lub tsev qiv ntawv loj | Cov ntaub ntawv theoreticalYield (ib Cell) | Ib leeg-baseQhov tseeb | Daim ntawv thov |
| CLR | 20 Kb, 30 Kb, thiab lwm yam. | 80 Gb rau 130 Gb | Kwv yees li.85% | De nov, SV hu, thiab lwm yam. |
| CCS | 15-20 Kb | 14 mus rau 40 Gb / Cell (Sequel II) 70 mus rau 110 Gb / Cell (Revio) Nyob ntawm cov qauv | Kwv yees li.99% | De nov, SNP/Indel/SV hu, Iso-Seq, |
Kev sib piv ntawm kev ua tau zoo thiab cov yam ntxwv ntawm Revio thiab Sequel II
| Cov ntsiab lus | Sequel II system | Revio qhov system | Nce |
| Qhov ceev dua | 8 million ZMWs | 25 million ZMWs | 3x |
| Kev ywj pheej theem | 1 | 4 | 4x |
| Shorter lub sij hawm khiav | 30 teev | 24 teev | 1.25x ib |
| 30X HiFi tib neeg genomes / xyoo | 88 | 1,300 ib | 15 x zwb |
Kev Pabcuam Zoo
● Tshaj li 8 xyoo dhau los ntawm PacBio sequencing platform nrog ntau txhiab qhov haujlwm kaw nrog ntau hom.
● Ua tiav nruab nrog qhov tseeb PacBio Sequencing platforms, Revio kom lav qhov kev sib tw txaus.
● Lub sijhawm tig rov qab sai dua, cov ntaub ntawv ntau dua thiab cov ntaub ntawv raug dua.
● Pab txhawb hauv ntau pua ntawm PacBio-raws li kev tshaj tawm uas cuam tshuam loj heev.
Cov Qauv Xav Tau
| Hom qauv | Tus nqi | Concentration (Qubit®) | Ntim | Purity | Lwm tus |
| Genomic DNA | Nyob ntawm cov ntaub ntawv xav tau | ≥50 ng / μl | ≥15μl | OD260/280 = 1.7-2.2; OD 260/230 = 1.8-2.5 hli Clear ncov ntawm 260 nm,tsis muaj kab mob | Concentration yuav tsum tau ntsuas los ntawm Qubit thiab Qubit/Nanopore = 0.8-2.5 |
| Total RNA | ≥1.2 μg | ≥120 ng / μl | ≥15μl | OD260/280 = 1.7-2.5; OD 260/230 = 0.5-2.5 hlitsis muaj kab mob | RIN tus nqi ≥7.5 5 ≥ 28S / 18S≥1 |
Kev Pabcuam Ua Haujlwm
Kev npaj ua qauv
Kev tsim kho tsev qiv ntawv
Sequencing
Cov ntaub ntawv tsom xam
Qhov project xa
1.In-house Data Yield
Cov ntaub ntawv tsim tawm los ntawm 63 CCS hlwb (los ntawm 26 hom)
| DATAPacBio-CCS-15 Kb | Nruab nrab | Max | Min | Median |
| Yield - subreads (Gb) | 421.12 Nws | 544.27 ib | 221.38 Nws | 426.58 ib |
| Yiled - CCS(Gb) | 25.93 Nws | 38.59 Nws | 10.86 Nws | 25.43 Nws |
| Polymerase N50 | 145,651 ib | 175,430 ib | 118, 118 ib | 144,689 ib |
| Cov ntaub ntawv N50 | 17,509 ib | 23,924 ib | 12.485 Nws | 17,584 ib |
| CCS N50 | 14.490 Nws | 19,034 ib | 9,876 ib | 14,747 ib |
| Qhov nruab nrab ntev-Polymerase | 67,995 ib | 89,379 ib | 49,664 ib | 66,433 ib |
| Nruab nrab ntev-Subreads | 15, 866 ib | 21,036 ib | 11,657 ib | 16,012 ib |
| Qhov nruab nrab ntev-CCS | 14,489 ib | 19,074 ib | 8,575 ib | 14,655 ib |
Cov ntaub ntawv tsim tawm los ntawm 16 CLR hlwb (los ntawm 76 hom)
| DATA-PacBio-CLR-30Kb | Nruab nrab | Max | Min | Median |
| Yield - subreads (Gb) | 142.20 Nws | 291.40 Nws | 50.55 Nws | 142.49 Nws |
| Polymerase N50 | 39,456 ib | 121, 191 ib | 15, 389 ib | 35,231 ib |
| Cov ntaub ntawv N50 | 28.490 Nws | 41,012 ib | 14.430 Nws | 29,063 ib |
| Qhov nruab nrab ntev-Polymerase | 22,063 ib | 48,886 ib | 8,747 ib | 21.555 Nws |
| Nruab nrab ntev-Subreads | 17.720 Nws | 27, 225 Ib | 8,293 ib | 17,779 ib |
2.Data QC – DemoStatistics ntawm cov ntaub ntawv tawm los
| Qauv | ccs Nyeem Num | Tag nrho ccs Bases (bp) | ccs Nyeem N50 (bp) | ccs Qhov nruab nrab Length (bp) | ccs Nyeem ntawv ntev tshaj plaws (bp) | Subread Bases (bp) | ccs tus nqi (%) |
| PB_BMKxxx | 3, 444, 159 ib | 54, 164, 122, 586 | 15,728 ib | 15,726 ib | 36, 110 Ib | 863,326,330,465 | 6.27 ib |
