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Plant Isoform-sequencing without Reference Genome

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Introductions

Isoform-sequencing without Reference Genome

PacBio's third-generation sequencing (Isoform-sequencing) uses single-molecule real-time sequencing technology (SMRT). Due to its ultra-long read length (average 15kb), complete full-length transcripts can be obtained directly without splicing, so higher quality transcripts can be obtained, which is conducive to the study of mRNA structure, such as variable splicing and fusion genes, Allele expression, etc. Overcome the shortcomings of the second-generation sequencing technology due to the short reads length. At present, full-length transcriptome sequencing technology has been widely used in plant research, including Arabidopsis, corn, wheat, sorghum, salvia, petunia, etc. Full-length transcriptome sequencing without reference genomes can be used to study the acquisition of more complete reference coding sequences of species without reference genomes, to study changes in variable splicing events caused by different experimental treatments, and more accurate quantitative analysis, etc.

Services

Based on PacBio's third-generation sequencing platform, Lifeasible uses single molecule real-time sequencing technology (SMRT) to provide professional isoform sequencing and analysis services without reference genome to help your research.

Workflow of isoform sequencing and analysis services without reference genome

Workflow of isoform sequencing and analysis services without reference genome

Analysis content

Standard infromation analysis Statistics and quality control of raw sequencing data
Full-length isoform data statistics
Basic function annotation of genes
a) Nr annotation
b) GO function annotation
c) COG/KOG annotatio
d) KEGG metabolic pathway annotation
e) SwissProt protein annotation
Advanced functional annotation of genes
a) Predicted coding protein frame(CDS)
b) Transcription factor analysis
c) R gene analysis (plants)
d) Protein domain analysis(Pfam, SMART)
e) TMHMM transmembrane spiral structure prediction
f) Signal peptide structure prediction
g) Protein O-GlcNAc glycosylation site prediction(mammalian)
h) ProP furin cleavage site prediction (eukaryote)
Structural analysis
a) Tandem repeat unit detection(SSR)
b) InCRNA analysis
c) Alternative splicing analysis
Customized information analysis Correction of the second-generation data to the third-generation data(illumina data required)
Quantitative and differential expression analysis of genes(illumina data required)
Multi-omics association analysis (such as methylation, proteomics, miRNA)

Sample requirements

  • Sample size
  • Total RNA>12μg (2 libraries)

  • Sample purity
  • OD260/280≥1.8, OD260/230≥1.0. And there is a normal peak at 260nm.

  • RNA integrity
  • RIN≥8.0, 28S/18S≥1.3, the baseline of the spectrum is not raised, and the 5S peak is normal.

  • Please put the sample in a 1.5ml tube, indicate the sample name, concentration and preparation time on the tube, and use parafilm to seal the tube mouth.
  • Before shipping, fix all sample tubes in 50ml centrifuge tubes with caps and ship them on dry ice.

Project cycle

  • The entire project generally takes about 2 months.

Feedback to customers

  • Raw data
  • A project report includes experimental process, instrument parameters, analysis methods and results, etc.

Service process

Service process of isoform sequencing and analysis services without reference genome

Advantages

  • Lifeasible specializes in providing services related to agricultural molecular breeding, with standardized systems and scientific management.
  • PacBio's third-generation sequencer has the advantages of ultra-long read length, high throughput, no GC preference, and direct detection of base modifications, etc.
  • The one-stop service we provide makes your upstream and downstream experiments seamlessly connected, facilitates your experiments more conveniently and quickly.
  • The continuously updated and expanded high-performance computing platform ensures efficient data processing and secure data storage.
  • Personalized customized package service.

※ It should be noted that our service is only used for research.

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