mRNA Sequencing

Nucleome Informatics is leading Transcriptome sequencing or RNA Sequencing (RNA-Seq) service provider in India. One of the most popular forms of transcriptome sequencing is mRNA-Seq, which targets all polyadenylated mRNA transcripts, or the coding portion of the transcriptome. The depth offered by next generation sequencing is leveraged to find new genes that were undetectable before due to their low level of expression. By the same token, the increased depth and reduced cost of sequencing means these projects can be used to profile gene expression while differentiating between isoforms of the same gene via paired-end reads.

 

Transcriptome sequencing with longer read length enables the identification of novel transcripts, alternative splicing and gene fusions, and scientists can identify biomarkers or key regulated genes, key functional genes related to different phenotypes, as well as key factors in temporal changes. RNA-Seq quantification with shorter read length can simultaneously measure the expression levels of many transcripts. It is widely used in disease research, drug response research, pharmacokinetics, and personalized healthcare research. We have published multiple publications using mRNA Seq application in high impact factor journals.

Small RNA Sequencing

Team Nucleome Informatics is very well regarded for its expertise in Small RNA sequencing and data analysis services in India. Small noncoding RNAs act in gene silencing and post-transcriptional regulation of gene expression. Small RNA sequencing (RNA-Seq) is a technique to isolate and sequence small RNA species, such as microRNAs (miRNAs). We frequently use Illumina sequencing technology for Small RNA-Seq to query thousands of small RNA and miRNA sequences with unprecedented sensitivity and dynamic range.

 

With small RNA-Seq you can discover novel miRNAs and other small noncoding RNAs, and examine the differential expression of all small RNAs in any sample. Using our services one of our clients has recently published a paper ‘Analysis of high iron rice lines reveals new miRNAs that target iron transporters in root’ in Journal of Experimental Botany where team Nucleome discovered 153 known and 41 novel miRNAs in roots of rice. You can use our service to characterize variations such as isomiRs with single-base resolution, as well as analyze any small RNA or miRNA without prior sequence or secondary structure information.

 

Advantage of Small RNA Sequencing at Nucleome:

 

Team Nucleome generates miRNA sequencing libraries directly from total RNA to understand the role of noncoding RNA. Our trained team of genomics and bioinformatics can help you to-

  • Understand how post-transcriptional regulation contributes to phenotype.
  • Identify novel biomarkers.
  • Capture the complete range of small RNA and miRNA species.

Whole Transcriptome Sequencing

Whole-transcriptome analysis with total RNA sequencing (RNA-Seq) detects coding plus multiple forms of noncoding RNA. Total RNA-Seq can accurately measure gene and transcript abundance, and identify known and novel features of the transcriptome. Total RNA-Seq provides optimal coverage in normal or low-quality samples. Species-specific ribosomal RNA probes (Ribo-Zero chemistry) can efficiently remove abundant RNA species. This leaves both fragmented and intact transcripts of interest for library capture.

 

We can also use this approach to capture long non coding RNA sequence (lncRNA Seq). Long non-coding RNAs (LncRNAs) are transcribed RNAs longer than 200 nt that do not encode for proteins. LncRNAs are involved in a wide range of important cellular processes such as X-chromosome inactivation, imprinting, maintenance of pluripotency, lineage commitment and apoptosis. LncRNAs are also known to be involved with human diseases such as cancer, cardiovascular disease and neurological disorders, and are of significant interest to researchers.

 

Nucleome bioinformatics team offers comprehensive analysis for both lncRNAs and mRNAs, enabling access to lncRNA and mRNA information in a single sequencing run. Applications include comparison of lncRNA and mRNA expressions in different stages of development and in different tissues, as well as unveiling key functions of mRNAs and lncRNAs.

Single Cell RNA Sequencing

Single-cell sequencing is now readily available in India to researchers through Nucleome’s expertise in this field. Nucleome is one of the few NGS providers with extensive experience in single cell sequencing technology, including single-cell RNA-Seq. With ultra-low-input and single-cell RNA-Seq, you can explore the distinct biology of individual cells within a complex tissue, and understand subpopulation responses to environmental cues. These assays enhance the study of cell function and heterogeneity in time-dependent processes such as differentiation, proliferation, and tumorigenesis.

 

Single-cell RNA-Seq enables the high-resolution transcriptome profiling of a single cell, and has broad utility for investigating developmental processes and gene regulatory networks, and for revealing heterogeneous gene expression patterns within cell cultures, tissues, and organs.

circularRNA Sequencing

Nucleome Informatics is a leading service provider of circular RNA Sequencing in India. The circRNA digitalization analysis based on HiSeq high-throughput sequencing uses the SBS-sequencing by synthesis, which can decrease the loss of nucleotides caused by the secondary structure. It is also powerful for its small requirement of sample quantity, high through-put, high accuracy with easily operated automatic platform.

 Sample requirement

  • Total RNA amount: ≥ 2.0 μg; RNA concentration: ≥ 50 ng/μl (For human and mouse, 200 ng could be accepted with risk.)
  • RIN value ≥ 6.3 for plants and fungi; RIN value ≥ 6.8 for animals
  • OD260/280 ≥ 2.0, OD260/230 ≥ 2.0, without degradation and DNA contamination

Recommended Sequencing Depth

  • ≥ 20 M reads per sample of 2x150bp on HiSeq 2500

 Sequencing Workflow:

The experimental procedure of circRNA sequencing is shown below:

Deliverables:

Option 1: Without Bioinformatics Analysis

  • Sample QC report
  • Raw Data in .fastQ format
  • Raw data Stats
  • Clean data QC
  • Sequencing and raw data QC report

Option 2: With Bioinformatics Analysis

  • Sample QC report
  • Raw Data in .fastQ format
  • Raw data Stats
  • Clean data QC
  • Sequencing and raw data QC report
  • Alignment of clean reads over reference genome
  • circRNA Identification, Quantification and DGE
  • circRNA source gene analysis
  • GO enrichment analysis
  • KEGG enrichment analysis
  • miRNA Binding site analysis
  • Tissue specific analysis

TAT: 3 to 4 months from the date samples pass QC