Plena Longitudo variant Sequencing-Nanopore
Servitium Commoda
Low sequence bias
Revelatio plena longitudo cDNA moleculis
● Minus notitia requiritur ad operiendum totidem transcripts
● Lepidium sativum multiple isoforms per gene
Expressio quantitatis in gradu isoform
Service Specifications
| Bibliotheca | Platform | Commendatur data cede (Gb) | Regimen quālitātis |
| cDNA-PCR (Poly-A ditata) | Nanopore PromethION P48 | VI Mb / sample (Prout species) | Plena longitudo ratio LXX% Mediocris qualitas score: Q10
|
Bioinformatics analyses
●Rudis MGE
Transumptum identificatio
Alternativum splicing
Expressio quantitatis in gene gradu et gradu isoform
Analysis differentialis expressio
Function annotationem et locupletationem (DEGs and DETS)
Sample Requisita et Delivery
Sample Requisita:
Nucleotides:
| Conc. | Moles (μg) | Puritas | Integritas |
| ≥ 100 | ≥ 0.6 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Nullam dapibus vel dapibus vel DNA contagione gel ostensum est. | Pro plantis: RIN≥7.0; Pro animalibus: RIN≥7.5; 5.0≥28S/18S≥1.0; limitata vel non collocantur elevatione |
TEXTUS: Pondus (siccus): ≥1 g
*Pro texti minore quam 5 mg, commendamus ut mico congelato (in liquido nitrogen) texti specimen mittat.
Cellam suspensionem: Cell comitem = 3×106- 1×107
* Commendamus navem gelidam cellam lysatam.In casu autem cellula minor quam V × 105mico constringitur in liquido nitrogenio commendatur, quod extractione microform potior est.
Exempla sanguinis: Volume≥1 ml
Sample Delivery commendatur
Continens: 2 ml tubum centrifugium (foil plumbi non commendatur)
Sample labeling: Group+replicata eg A1, A2, A3;B1, B2, B3...
Shipment: 2、 siccum-glacies: Exemplaria in saccis referta necesse est et in siccitate glaciei sepulta.
- RNAstable tube: RNA specimina in RNA stabilizationis tubo exsiccata possunt (eg RNAstable®) et in cella temperie conscendunt.
Service Opus O
Nucleotides:
Sample traditio
Library construction
Sequencing
Analysis
Post-venditionis officia
Service Opus O
TEXTUS:
Experimentum design
Sample traditio
RNA extractionem
Library construction
Sequencing
Analysis
Post-venditionis officia
1.Analysis differentialis expressionis -Volcano insidiarum
Analysis differentialis expressionis in utroque gradu gene discurrere potest ad cognoscendum differentialiter genesis (DEGs) expressum et in gradu isoformi ad differentiam cognoscendam.
transcripts expressit (DEts)
2.pampineis hierarchicis heatmap
3.Alternative splicing idem et divisio
Quinque genera rerum discurrentia ab Astalavista praedici possunt.
4.Alternative poly-adienylation (APA) eventuum agnitio et ARGUMENTUM apud 50 bp flumine poly-A.
BMK Case
Alternative splicing identification and isoform-level quantitas per nanopore plena longitudinis transcriptum.
Published:Natura Communicationum, 2020
De consilio sequendo:
Ordinationem: 1. CLL-SF3B1(WT);2. CLL-SF3B1(K700E mutationis);3. Normal B-cellulae
Sequentia deerant: MinION 2D bibliotheca sequencing, PromethION 1D bibliotheca sequencing;brevis-legere notitia ex eodem exempla
Sequentia suggestum: Nanopore Minion;Nanopore PromethION;
Eventus key
1.Isoform-gradu Alternativus Splicing Lepidium sativum
Longum-legere sequentia facit idem ex mutant SF3B1K700E-altered splice sites ad isoform-gradu.35 alternative 3′SSs et 10 alternative 5′SSs inventae sunt signanter differentialiter divisae inter SF3B1K700Eet SF3B1WT.33 de 35 alteratione nuper inventae sunt per sequentia longitudinis lectionis.
2.Isoform-gradu Alternativus Splicing quantitatis
Expressio retentionis intron(IR) isoforms in SF3B1K700Eet SF3B1WTquanta secundum sequentia nanopore erant, ostendens global down-ordinationem IR isoforms in SF3B1K700E.
Reference
Tang AD , Soulette CM , Baren MJV , et al.Translatio plena-length characterizationis mutationis SF3B1 in chronicis lymphocyticis leukemia indicat downregulation of introns retentis [J].Naturae Communicationis.
