Next-Generation Sequencing

Bioserve is not only about providing standard eukaryotic RNA sequencing services. We are also dedicated to providing prokaryotic RNA sequencing services using the latest techniques to meet your bacterial gene expression profiling needs.In recent years, high throughput sequencing of cDNA libraries (RNA-Seq) has emerged as a powerful technology for profiling gene expression. It helps to identify novel transcripts, discover previously unannotated genes, and map transcriptome architecture in a wide variety of bacterial species. With the application of RNA-seq derivative approaches, we can gain biological insights into the bacterial world and aspire to uncover the mysteries behind gene expression, organization, and other functional genomic features. Bacterial transcriptome and metatranscriptome information is important for predicting resistance to specific antibiotics, understanding host-pathogen immune interactions, quantifying gene expression changes, and tracking disease progression.There are obvious differences in cDNA library construction between bacterial RNA-Seq and Eukaryotic RNA-Seq. Bacterial RNA-Seq workflow’s first step is the selection of mRNA transcripts. Ribo-Zero/Ribo-minus ribosomal RNA reduction chemistry is used in place of a poly tail. A tail selection which makes this method particularly suited for bacteria mRNA is a poly-A tail. Following its purification, the mRNA is fragmented into small pieces, and copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity is achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM), followed by second strand cDNA synthesis. Through the use of strand-specific RNA-Seq, a more complete understanding of the transcriptome could be achieved. This has the potential to identify new levels of regulation in gene expression.

Bioserve is offering high-throughput and cost-efficient lncRNA sequencing service for human, mouse, rat and plant genomes by combining the latest Illumina sequencing instrument and advanced bioinformatics analysis. Bioserve’s bioinformatics team, composed entirely of PhDs, provides a comprehensive analysis for both lncRNAs and mRNAs, enabling access to lncRNA and mRNA information in a single sequencing run. Applications include a comparison of lncRNA and mRNA expressions in different development stages and different tissues, as well as unveiling key functions of mRNAs and lncRNAs.

lncRNAs are defined as a large and diverse class of transcribed RNAs, of sizes greater than 200 nt, that do not encode proteins. LncRNAs are widely distributed in organisms and lncRNA transcripts account for a major part of the non-coding transcriptome. LncRNAs may be classified into different subtypes (including antisenses, intergenic, overlapping, intronic, bidirectional, and processed) based on the position and direction of transcription in relation to other genes. LncRNAs resemble mRNAs because they are typically transcribed from active chromatin, polyadenylated, and capped. However, they do not direct protein synthesis.

The application of next-generation sequencing technology has greatly facilitated the discovery and function analysis of lncRNAs. LncRNA sequence information can be acquired at single-base resolution via library preparation, high-throughput sequencing, and powerful bioinformatics analysis. We construct the sequencing library by the removal of rRNA and retain both lncRNAs and mRNAs. The lncRNA-mRNA interaction analysis contributes to the illumination of lncRNA regulatory networks.

To support the increasing research interest in small RNA, Bioserve is offering a qualified small RNA sequencing service to analyze novel miRNA and other small RNA species. This service offers novel small RNA discovery, mutation characterization, and expression profiling of small RNAs by leveraging of advanced NGS technologies and data analysis pipeline.

Small RNA species generally include the most common and well-studied microRNA, small interfering RNA (siRNA), and piwi-interacting RNA (piRNA), and also other types of small RNA, such as small nucleolar RNA (snoRNA) and small nuclear RNA (snRNA). Small RNA is a type of lowly abundant, short in length (<200 nt), non-protein-coding RNAs that lack polyadenylation. Small RNA populations can vary significantly among different tissue types and species. Generally, small RNAs are formed by fragmentation of longer RNA sequences with the help of dedicated sets of enzymes and other proteins.

Small RNAs act in gene silencing and post-transcriptional regulation of gene expression. However, small RNA is not sufficient for the induction of RNA inference. It generally needs to form the core of the RNA-protein complex known as RNA-induced silencing complex (RISC). siRNAs can cleave the mRNA in the middle of the mRNA-siRNA duplex, and the resulting mRNA halves are degraded by other cellular enzymes. Unlike the siRNA pathway, miRNA-mediated degradation is initiated by enzymatic removal of the mRNA polyA tail. piRNAs are essential for the development of germ cells. Small RNAs have been demonstrated to be involved in a number of biological processes including development, cell proliferation and differentiation, and apoptosis.

NGS offers tremendous output with unprecedented sensitivity and dynamic range and can identify weakly expressed small RNAs as well as quantitatively reveal heterogeneity in length and sequence. It’s a powerful tool for investigating the function of small RNAs and prediction of potential mRNA target molecules without requiring available reference genomes. Obtaining a premium small RNA sequencing library begins with the isolation of small RNAs by size fractionation using gel electrophoresis selection or silica spin columns from total RNA. Following RNA adapter ligation using a 5’ adenylated DNA adapter with a blocked 3’end, small RNAs are reverse transcribed, amplified by PCR and sequenced. To identify and annotate known miRNAs, the sequencing reads can be mapped to a species-specific database, such as miRWalk and miRBase.