DNA / RNA sequencing -PacBio Sequencer
Likaroloana
Mekhoa e 'meli ea tatellano ea sequencer ea PacBio: Continuous Long Read (CLR) le Circular Consensus Read (CCS)
| Sequencing Mode | Boholo ba Laebrari | Lintlha tsa TheoryYield (Per Cell) | Motheo o le mongHo nepahala | Lisebelisoa |
| CLR | 20Kb, 30Kb, joalo-joalo. | 80 Gb ho isa ho 130 Gb | Hoo e ka bang.85% | De novo, ho letsetsa SV, joalo-joalo. |
| CCS | 15-20 KB | 14 ho isa ho 40 Gb/Cell (Sequel II) 70 ho isa ho 110 Gb/Cell (Revio) E itšetlehile ka mehlala | Hoo e ka bang.99% | De novo, SNP/Indel/SV letsa, Iso-Seq, |
Papiso ea ts'ebetso le likarolo tsa Revio le Sequel II
| Melao | Sistimi ea sequel II | Sistimi ea Revio | Nyollelo |
| Boima bo phahameng | Limilione tse 8 tsa ZMW | Li-ZMW tse limilione tse 25 | 3x |
| Mekhahlelo e ikemetseng | 1 | 4 | 4x |
| Linako tse khutšoane tsa ho matha | Lihora tse 30 | Lihora tse 24 | 1.25x |
| 30X HiFi liphatsa tsa lefutso / selemo | 88 | 1,300 | 15x ka kakaretso |
Melemo ea Tšebeletso
● Boiphihlelo ba lilemo tse fetang 8 sethaleng sa tatellano sa PacBio le likete tsa merero e koetsoeng e nang le mefuta e fapaneng.
● E na le lisebelisoa tsa morao-rao tsa PacBio Sequencing, Revio ho netefatsa hore ho na le tatellano e lekaneng.
● Nako ea ho fetola kapele, tlhahiso e phahameng ea data le lintlha tse nepahetseng haholoanyane.
● E kentsoe likhatisong tse makholo tse nang le tšusumetso e phahameng ea PacBio.
Litlhoko tsa Mohlala
| Mofuta oa Mohlala | Chelete | Concentration (Qubit®) | Bolumo | Bohloeki | Ba bang |
| Genomic DNA | Itšetlehile ka Tlhokahalo ea Lintlha | ≥50 ng/μl | ≥15μl | OD260/280=1.7-2.2; OD260/230=1.8-2.5; Tlhōrō e hlakileng ho 260 nm,ha ho na litšila | Tekanyo e lokela ho lekanyetsoa ka Qubit le Qubit/Nanopore = 0.8-2.5 |
| Kakaretso ea RNA | ≥1.2μg | ≥120 ng/μl | ≥15μl | OD260/280=1.7-2.5; OD260/230=0.5-2.5;ha ho na litšila | RIN boleng ≥7.5 5≥28S/18S≥1 |
Ts'ebetso ea ts'ebetso ea litšebeletso
Ho lokisetsa mohlala
Kaho ea laebrari
Tatelano
Tlhahlobo ea lintlha
Phano ea morero
1.Tlhahiso ea Boitsebiso ka tlung
Lintlha tse hlahisoang ho tsoa liseleng tse 63 tsa CCS (ho tsoa ho mefuta e 26)
| DATA-PacBio-CCS-15 KB | Karolelano | Max | Min | Median |
| Lihlahisoa - li-subreads (Gb) | 421.12 | 544.27 | 221.38 | 426.58 |
| Yeled - CCS(Gb) | 25.93 | 38.59 | 10.86 | 25.43 |
| Polymerase N50 | 145,651 | 175,430 | 118,118 | 144,689 |
| Subreads N50 | 17,509 | 23,924 | 12,485 | 17,584 |
| CCS N50 | 14,490 | 19,034 | 9,876 | 14,747 |
| Karolelano ea bolelele-Polymerase | 67,995 | 89,379 | 49,664 | 66,433 |
| Karolelano ea bolelele-Makaroloana | 15,866 | 21,036 | 11,657 | 16,012 |
| Karolelano ea bolelele-CCS | 14,489 | 19,074 | 8,575 | 14,655 |
Lintlha tse hlahisoang ho tsoa liseleng tse 16 tsa CLR (ho tsoa ho mefuta e 76)
| DATA-PacBio-CLR-30Kb | Karolelano | Max | Min | Median |
| Lihlahisoa - li-subreads (Gb) | 142.20 | 291.40 | 50.55 | 142.49 |
| Polymerase N50 | 39,456 | 121,191 | 15,389 | 35,231 |
| Subreads N50 | 28,490 | 41,012 | 14,430 | 29,063 |
| Karolelano ea bolelele-Polymerase | 22,063 | 48,886 | 8,747 | 21,555 |
| Karolelano ea bolelele-Makaroloana | 17,720 | 27,225 | 8,293 | 17,779 |
2.Data QC - DemoLipalopalo tsa tlhahiso ea data
| Mohlala | ccs Bala Num | Kakaretso ea Metheo ea ccs (bp) | ccs Bala N50 (bp) | ccs Mean Length (bp) | ccs Longest Bala (bp) | Subreads Bases (bp) | sekhahla sa ccs(%) |
| PB_BMKxxx | 3,444,159 | 54,164,122,586 | 15,728 | 15,726 | 36,110 | 863,326,330,465 | 6.27 |
