Plant Y1H/Y2H Positive Clone Sequencing and Hit Analysis Service
Post-screening Identification and Annotation
CD BioSciences provides positive clone sequencing and hit analysis services for plant Y1H and Y2H screening projects. The service supports the identification, organization, and preliminary interpretation of candidate prey inserts recovered from yeast hybrid screens.
After positive colonies are obtained, sequencing and annotation are needed to determine which genes or protein fragments were enriched, remove redundant clones, and prioritize candidates for point-to-point validation or downstream functional assays.
Service Overview
Positive colonies from Y1H or Y2H screening can contain true interactors, repeated clones, partial inserts, mixed colonies, or background-derived candidates. A structured post-screening workflow helps convert colony growth results into a usable candidate list.
Depending on the project scale and screening design, positive clone analysis can include colony re-streaking, PCR confirmation, insert size checking, plasmid rescue, Sanger sequencing, next-generation sequencing support, sequence trimming, database alignment, gene annotation, and candidate table preparation.
Positive Clone Processing Workflow
Figure 1. Positive clone processing workflow from colony picking to sequence-based candidate identification.
- Positive colony picking and secondary selection
- Colony PCR or insert amplification
- Insert size assessment
- Prey plasmid extraction or rescue, when required
- Sanger sequencing or sequencing library preparation
- Sequence quality review and trimming
- Database alignment and gene annotation
- Redundancy removal and candidate hit table preparation
Analysis Content
Figure 2. Example information structure for yeast hybrid hit analysis deliverables.
| Analysis Item | Purpose |
|---|---|
| Sequence quality review | Checks read quality, vector sequence, ambiguous bases, and usable insert length. |
| Database alignment | Identifies candidate genes or transcript fragments by similarity search against suitable reference databases. |
| Redundancy removal | Groups repeated clones and recurring genes to support candidate prioritization. |
| Functional annotation | Summarizes gene ID, predicted function, protein family, domain information, or GO/KEGG terms when available. |
| Validation priority notes | Flags candidates suitable for point-to-point Y1H/Y2H validation or orthogonal assays. |
Sequencing Options
Suitable for standard positive clone identification when the number of colonies is moderate and individual clone traceability is needed.
Can be considered when many positive colonies are obtained or when broader representation of enriched prey inserts is required.
Maintains colony-to-sequence correspondence for candidate confirmation and follow-up validation planning.
Converts sequencing results into a structured hit list with gene identifiers, descriptions, and prioritization notes.
Quality Considerations
Sequencing results should be interpreted together with colony phenotype, selection stringency, bait auto-activation background, and validation design. A frequently recovered hit may be biologically relevant, but it may also reflect library representation, prey expression advantage, or non-specific yeast growth.
| Issue | How It Is Addressed |
|---|---|
| Mixed colony or multiple inserts | Secondary selection, colony purification, PCR review, or re-sequencing may be recommended. |
| Short or low-quality reads | Sequence trimming, primer review, repeat sequencing, or alternative identification workflow may be used. |
| Redundant clones | Repeated hits are grouped to avoid overcounting during candidate prioritization. |
| False-positive candidates | Point-to-point validation and independent assays are recommended for selected hits. |
Sample and Information Requirements
- Positive yeast colonies or preserved positive clone material
- Assay type: Y1H, nuclear Y2H, or membrane Y2H
- Bait information and screening selection conditions
- Prey vector information and sequencing primer information, if available
- Plant species and preferred reference database
- Expected sequencing depth or number of clones for analysis
Deliverables
- Positive clone processing records
- PCR or insert size images, when included
- Sequencing files or sequencing result summaries
- Cleaned sequence information
- Candidate gene or protein annotation table
- Redundancy and hit frequency summary
- Validation recommendation notes
- Final report with raw data appendix
Related Services
For positive clone sequencing and hit analysis, please contact us with the assay type, bait information, and expected number of colonies for analysis.
For research use only, not for clinical use.