Molecular Services / Next-Generation Sequencing

Next-Generation Sequencing (NGS) is massively parallel, per run simultaneously sequencing millions of fragments. This high-throughput approach converts at one time into hundreds to thousands of genes being sequenced. NGS also provides greater discovery ability with deep sequencing to identify novel or unusual variants. Bioserve offers an end-to-end solution for Next-Generation Sequencing as a service for a wide range of sample types including DNA and RNA. It uses multiple sequencing platforms including Illumina NextSeq 500, HiSeq, MiSeq, PacBio Sequel, and Oxford Nanopore Technology to achieve this. Our team of scientists and Bioinformaticians work with you to deliver reliable high-quality results from any type of source with limited nucleic acids. We provide end-to-end solutions for your NGS project needs.

  • Epigenomics
  • Genomic Sequencing
  • Microbial Genomics
  • Transcriptomics
  • ChIP Sequencing
  • MeDIP Sequencing
  • Whole Genome Bisulphate Sequencing

Chromatin Immunoprecipitation (ChIP) is a classic experiment method to study the interactions between proteins and DNA. The combination of ChIP with high-throughput sequencing (ChIP-seq) can accurately screen and determine the protein binding sites in the whole genome, so it is widely applied to the research on such related fields as histone modifications and transcription factor regulation, etc.

Bioserve is dedicated to producing high-quality ChIP-seq data sets to help you profiling DNA targets for histone modification, structural proteins, transcription factors, and other DNA-associated proteins at a genome-wide scale.

Applications:

  • Gene regulation
  • Transcription complex assembly
  • DNA repair
  • Histone modification
  • Developmental mechanisms
  • Disease progression

Service Specification

Sample requirements and preparation
  • Sample type: ChIPed-DNA
  • DNA amount: ≥ 50 ng, DNA concentration: ≥ 10 ng/µl, DNA volume: ≥ 10 μl
  • OD260/280 = 1.8~2.0 without degradation or RNA contamination
  • 200-300-bp insert bisulfite treated DNA library
Sequencing
  • Illumina NextSeq 500/ HiSeq Platforms
  • Paired-end 150 bp
  • More than 80% of bases with a ≥Q30 quality score
  • ≥10M clean reads (transcription factor), ≥20 M clean reads (histone proteins)

 

Bioinformatics Analysis

Basic Analysis
  • Quality control
  • Read mapping
  • Peak calling
  • Differential analysis
  • Joint analysis (in case of large number of samples)
  • Identification of binding sites
Advanced Analysis
  • Basic +
  • Motif analysis
  • GO analysis
  • Publication
Turn-around Time:

6 weeks to deliver the raw data plus 4-5 weeks for analysis depends on the data volume and aim of the study.

Data Storage:

6 months

Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) or DNA immunoprecipitation sequencing (DIP-Seq) is commonly used to study 5mC or 5hmC modification. Specific antibodies can be used to study cytosine modifications. If using 5mC-specific antibodies, methylated DNA is isolated from genomic DNA via immunoprecipitation. Anti-5mC antibodies are incubated with fragmented genomic DNA and precipitated, followed by DNA purification and sequencing. Deep sequencing provides greater genome coverage, representing the majority of immunoprecipitated methylated DNA.

Pros:

  • Covers CpG and non-CpG 5mC throughout the genome
  • 5mC in dense, less dense, and repeat regions are covered
  • Antibody-based selection is independent of sequence and does not enrich for 5hmC due to antibody specificity

Cons:

  • Base-pair resolution is lower (~150 bp) as opposed to single base resolution
  • Antibody specificity and selectivity must be tested to avoid nonspecific interaction

Antibody-based selection is biased towards hypermethylated regions.

 

 

Service Specification

Sample requirements and preparation
  • Sample type: >5 uG of DNA without RNA contamination
  • 200 mg to 500 mg tissue
  • Samples sources including human, animals, plants and microorganisms
  • OD260/280=1.8~2.0
  • 250-300-bp insert bisulfite treated DNA library
Sequencing
  • Illumina NextSeq 500
  • Paired-end 150 bp
  • More than 80% of bases with a ≥Q30 quality score
  • Sequencing depth > 20X

 

Workflow

Bioinformatics Analysis

Basic Analysis
  • Quality filtration of reads,
  • Alignment of MeDIP seq reads to reference genome,
  • Mapping summary statistics,
  • Chromosomewise mapping stats
  • enriched regions-Peaks coordinates
  • Differentially methylated genes/region
  • Differentially methylated region annotation
Advanced Analysis
  • Basic+
  • Gene ontology of shortlisted genes (if available)
  • Shortlisted genes Pathway analysis (if available)
  • Publication support
Turn-around Time:

6 weeks to deliver the raw data plus 4-5 weeks for analysis depends on the data volume and aim of the study

Data Storage:

6 months

Whole Genome Bisulphate Sequencing

Whole genome bisulfite sequencing is an effective and reliable strategy to identify individually methylated cytosines on a genome-wide scale with a range of applications including studies on gene regulation, stem cell differentiation, embryogenesis, aging, cancer and other diseases, and phenotypic diversity and evolution in plants and animals. With over 15 years of experience and the state-of-the-art Genomics platforms Bioserve provides whole genome sequencing of bisulfite-converted DNA as an effective method to identify individually methylated cytosines on a genome-wide scale.  we can totally meet your project requirements and budgets in the exploration of methylome.

DNA methylation at the C5 position of cytosine plays a crucial role in gene expression and chromatin remodeling, and perturbations in methylation patterns are implicated in the development of cancer, neurodegenerative diseases, and neurological disorders. Therefore, the mapping of methylome is critical to understanding gene expression and other processes subject to epigenetic regulation.

 
Workflow

 

 

Service Specification

Sample requirements and preparation
  • Sample type: >5 uG of DNA without RNA contamination
  • 200 mg to 500 mg tissue
  • Samples sources including human, animals, plants and microorganisms
  • OD260/280=1.8~2.0
  • 250-300-bp insert bisulfite treated DNA library
Sequencing
  • Illumina NextSeq 500
  • Paired-end 150 bp
  • More than 80% of bases with a ≥Q30 quality score
  • Sequencing depth > 20X

 

Bioinformatics Analysis

Basic Analysis
  • Quality filtration of data,
  • Genome alignment to the insilico converted bisulfite genome,
  • Alignment files in .bam format,
  • Methylation density/Genome-wide methylation profile identification,
  • Differentially methylated Genes,
  • CpG track for visualization (if client ask for it)
  • Gene ontology of shortlisted genes (if available)
  • Shortlisted genes Pathway analysis (if available)
Advanced Analysis
  • Basic+
  • High-density plots
  • Publication support
Turn-around Time:

6 weeks to deliver the raw data plus 4-5 weeks for analysis depends on the data volume and aim of the study.

Data Storage:
6 months
  • Amplicon Sequencing
  • mtDNA Sequencing
  • Targeted Region Sequencing
  • Viral Genome Sequencing
  • Whole Exome Sequencing
  • Whole Genome Sequencing
Amplicon Sequencing

Amplicon sequencing is based on NGS technology. The ultra-deep sequencing of amplicons that include small PCR products or long range PCR products up to 30 kb allows efficient variant identification and characterization.  This technique has a wide range of applications, including 16S/18S/ITS gene sequencing, SNP genotyping, genome editing clone verification, somatic/complex variant discovery, and antibody heavy or light chain et al.

Whether you would like to detect the diversity of microbial communities or discover rare somatic mutations in complex samples. Bioserve could provide professional, cost-efficient and high-speed amplicon sequencing services to meet your project requirements.

Key Features and Advantages:
  • Providing high-sensitive detection levels through ultra-deep sequencing
  • Achieving high coverage by sequencing hundreds of thousands of amplicons per reaction
  • Cost-efficient and fast turnaround time
  • Microbial culture is not necessary
  • Wide range of amplicons from 100 bp to 10 kb
Sample Types:

Purified or unpurified PCR products, fragmented DNA, gDNA, restriction digested material, and plasmids

 
Work Flow:

Service Specifications:

Sample requirements and preparation
  • Sample Type
    • Purified genomic DNA
    • Frozen cell pellets (mammalian or bacterial)
    • PCR amplicons
    • Select tissues
    • Blood

 

  • DNA amount
    • >100 ng for Illumina PE library
Sequencing
  • Illumina Nextseq 500
  • Read Length 150 PE
  • Depth of coverage ≥ 1000x.
  • More than 80% of bases with a ≥Q30 quality score.

Bioinformatics Analysis

Basic Analysis
  • QC
  • Contamination screen
  • Mapping
  • Variant calling for SNVs only
  • Variant annotation with
Advanced Analysis: 
  • Basic +
  • Annotation with commercial dbs
  • Clincial effect prediction
  • Variant calling for SNVs, MNVs and SV s
  • Functional effect prediction
Turn-around Time:

4 weeks to deliver the raw data plus 4-5 weeks for analysis depends on the data volume.

mtDNA Sequencing

Bioserve offers mtDNA sequencing on Illumina platform. mtDNA is a compact, double-stranded circular genome of ~ 16.5 kb with a cytosine-rich light chain and a guanine-rich heavy chain. Mitochondria plays very important role in important cellular functions. The mutant mtDNA and the wild type may co-exist as heteroplasmy, and cause human disease, including cancer, heart disease, diabetes, Alzheimer’s disease, Parkinson’s disease and hypertension. Because there is considerable clinical variability between mitochondrial disorders and many patients who exhibit phenotypes that overlap diseases, often diagnosis may only be confirmed by identification of a pathogenic mtDNA variant through molecular genetic testing of DNA extracted from a blood sample. Mitochondrial DNA sequencing is a useful tool for researchers studying human diseases, and be also used in population genetics and biodiversity assessments.

Mitochondrial DNA sequencing is available on Illumina’s NextSeq. Illumina next-generation sequencing (NGS) has the potential to transform mtDNA analysis. Bioserve offers mtDNA sequencing service using long range PCR approach to amplify the mtDNA if the reference is available, otherwise isolating the mtDNA using commercially available kits followed by shotgun library preparation.

 
Work Flow

Service Specifications:

Sample requirements and preparation
  • DNA amount
    • >100 ng for Illumina PE library
Sequencing
  • Illumina Nextseq 500
  • Read Length 150 PE with 200 bp insert size
  • Depth of coverage ≥ 1000x.
  • More than 80% of bases with a ≥Q30 quality score.

Bioinformatics Analysis

Basic Analysis
  • QC
  • Contamination screen
  • Mapping
  • Variant calling for SNVs only
  • Variant annotation with OSS
Advanced Analysis
  • Basic +
  • Annotation with commercial dbs
  • Clincial effect prediction
  • Variant calling for SNVs, MNVs,CNVs and SV s
  • Functional effect prediction
  • Genealogy prediction
Turn-around Time:

3-4 weeks for sequencing plus 4-5 weeks for analysis depends on the data volume.

Data Storage:

6 months

Targeted Region Sequencing

Targeted resequencing is an effective method for analyzing the desired genes/SNPs/Regions of interest by next generation sequencing. Whether you are interested in a few genes, or a few hundred genes, targeted resequencing panels offer high sensitivity and specificity, providing in-depth coverage, resulting in high-quality data with maximum on targets covered across all sample types like EDTA, Plasma and FFPE.

With targeted resequencing gene panels, you can discover point mutations, insertions/deletions (INDELs), copy number variations (CNVs), and gene rearrangements.

We at Bioserve use Illumina, Agilent and Nimblegen enrichment methods for Illumina sequencing and Ampliseq method on Ion S5 sequencing. So far Bioserve has processed more than 2000 cancer samples using Agilent enrichment method.

Key Applications:
  • Discover genetic biomarkers associated with disease state
  • Characterize genetic associations to a particular phenotype, such as drug response
  • Characterize genetic associations to a particular phenotype, such as drug response
  • Design genetic tests, such as for pharmacogenomics or oncology
 
Work Flow

Service Specifications:

Sample requirements and preparation
  • Sample Type
    • Purified genomic DNA
    • Frozen cell pellets (mammalian or bacterial)
    • PCR amplicons
    • Formalin-fixed, paraffin-embedded (FFPE) thin sections (slides)
    • Select tissues
    • Blood

 

  • DNA amount
    • >100 ng for Illumina PE library
Sequencing
  • Illumina Nextseq 500 and Ion S5
  • Read Length 150 PE
  • Depth of coverage ≥ 1000x.
  • More than 80% of bases with a ≥Q30 quality score.

Bioinformatics Analysis

Basic Analysis
  • QC
  • Contamination screen
  • Mapping
  • Variant calling for SNVs only
  • Variant annotation with OSS
Advanced Analysis
  • Basic +
  • Annotation with commercial dbs
  • Clincial effect prediction
  • Variant calling for SNVs, MNVs,CNVs and SV s
  • Functional effect prediction
Turn-around Time:

7 weeks to receive the probes and 4 weeks to deliver the raw data plus 4-5 weeks for analysis depends on the data volume.

Data Storage:

6 months

  • No reference sequence required
  • Sequence With the Depth of Coverage that Makes Sense for Your Project
  • Complete characterization of viral genomes
  • Identify and quantify minor variants
  • Generate complete de novo assemblies of large viral genomes
  • Cost-effective high-throughput sequencing
  • High Quality Data& Fast Turnaround
 
Viral Sequencing work flow

Service Specifications:

Sample requirements and preparation
  • Viral DNA / cDNA amount
    • >500 ng for Illumina PE library
Sequencing
  • Illumina Nextseq 500
  • Read Length 150 PE with 200 bp insert size
  • Depth of coverage ≥ 1000x.
  • More than 80% of bases with a ≥Q30 quality score.

Bioinformatics Analysis

Basic Analysis
  • QC
  • Contamination screen
  • Mapping
  • Variant calling for SNVs only
  • Variant annotation with OSS
Advanced Analysis
  • Basic +
  • Annotation with commercial dbs
  • Clincial effect prediction
  • Variant calling for SNVs, MNVs,CNVs and SV s
  • Functional effect prediction
  • Phylogeny analysis
Turn-around Time:

3-4 weeks for sequencing plus 4-5 weeks for analysis depends on the data volume.

Data Storage:

6 months

Whole Exome Sequencing

Bioserve has been providing an affordable whole exome sequencing service since 2016 using Agilent and Illumina Exome enrichment approaches. We have vast experience in delivering high-quality data on FFPE derived DNA and cfDNA also. We employ Illumina NextSeq 500 sequencing platform to generate the raw data. Bioserve has completed more than 500 WES samples until now.

 
Whole exome sequencing work flow

Service Specifications:

Sample requirements and preparation
  • DNA amount
    • 2 uG for Agilent libraries and 500 ng for Illumina library
    • OD260/280=1.8~2.0.
  • No RNA contamination
  • All DNA samples are further validated for purity and quantity.
  • Library preparation using Illumina TruSeq Exome Kit / Agilent Sureselect XT All exons plus UTR kit
Sequencing
  • Illumina Nextseq 500
  • Read Length 150 PE with 300 bp insert size
  • Depth of coverage ≥ 100x.
  • More than 80% of bases with a ≥Q30 quality score.
  • Recommended coverage for Mendelian disorder/rare disease is 50X and 100X for tumor samples.

Bioinformatics Analysis

Basic Analysis
  • QC
  • Contamination screen
  • Mapping
  • Variant calling for SNVs only
  • Variant annotation with OSS
Advanced Analysis
  • Basic +
  • Annotation with commercial dbs
  • Clincial effect prediction
  • Variant calling for SNVs, MNVs and SV s
  • Functional effect prediction
  • Population statistics
  • Coding and non-coding variant annotation
Turn-around Time:

3-4 weeks for sequencing plus 4-5 weeks for analysis depends on the data volume.

Data Storage:

6 months

Whole Genome Sequencing

Whole Genome Sequencing (WGS) provides the most comprehensive map of an organism’s genetic make-up. Unlike targeted sequencing, WGS covers all corners of an organisms’ entire genome, which makes it possibly the most important of molecular data. This approach is a highly effective way to study a microbiome’s virulence, drug resistance or novel drug targeting. It is useful for the study of cancer and a variety of diseases, as well as human population evolution studies and pharmacogenomics. We can obtain high-resolution genomic variations is through resequencing or de novo assembly using comparative genomics for eukaryotes. It is now possible to generate high-quality sequencing data with as little as 10 ng of starting material. Bioserve is equipped with Illumina NextSeq 500, HiSeq, Miseq, and Ion S5 and is capable of simultaneously sequencing more than 500 WGS projects on its Illumina platform.

 
Whole Genome Sequencing Workflow:

Service Specifications:

 

Sample requirements and preparation
  • DNA amount
    • For shotgun libraries 500 ng to 1000 ng
    • For Matepair libraries 10 uG
    • OD260/280=1.8~2.0.
    • No RNA contamination
    • All DNA samples are further validated for purity and quantity.

 

 
Sequencing
  • Illumina Nextseq.
  • Read Length 150 PE with 300 bp insert size
  • Depth of coverage ≥ 100x.
  • More than 80% of bases with a ≥Q30 quality score.
  • Both paired-end library and mate-pair library can be constructed in the library preparation step.

 

Bioinformatics Analysis

We provide customized bioinformatics analysis including:

Basic Analysis
  • QC
  • Contamination screen
  • Mapping
  • Variant calling for SNVs only
  • Variant annotation with OSS
Advanced Analysis
  • Basic +
  • Annotation with commercial dbs
  • Clincial effect prediction
  • Variant calling for SNVs, MNVs and SV s
  • Functional effect prediction
  • Population statistics
  • Coding and non-coding variant annotation
Turn-around Time:

3-4 weeks for sequencing plus 4-5 weeks for analysis depends on the data volume

Data Storage:

6 Months

  • 16S/18S/ITS Amplicon Sequencing
  • Metagenomic Shotgun Sequencing
  • Metatranscriptome Sequencing
16S/18S/ITS Amplicon Sequencing

16s rRNA gene, a highly conserved component, is the most widely used gene marker for genus and species identification and taxonomic significance in bacteria and archaea. The estimated substitution rate for hypervariable regions is approximately 7000 times higher than the highly conserved ones, which contain abundant taxonomic information based on these genetic differences. This makes the 16S gene amplicons obtained from PCR useful to deduce taxonomic identifications based upon bioinformatics alignments. However, 18S rRNA is also commonly used in fungi for phylogenetics as it has more hypervariable domains than 16S. In addition to this, the ITS region, removed in the post-transcriptional process of nuclear rRNA cistron, is widely regarded as a universal fungi barcode marker for the successful identification of the broadest range of fungi. Compared to 18S, ITS is more variable and is more suitable as the genetic marker for measuring intraspecific genetic diversity.

16S/18S/ITS Amplicon Sequencing is now a well-established method for microbial identification and phylogeny studies of samples from complicated microbiomes or environments.

Advantages of 16S/18S/ITS Amplicon Sequencing include the following:

  • Speed, cost-efficiency, and high-precision.
  • Measures the most common housekeeping genetic markers with conserved and variable regions.
  • Suitable for multiple applications like microbial identification, diversity analysis, taxonomy and phylogeny, new species determination, relationship study of microorganism and disease, metagenomics, etc.
 
Workflow

Service Specification

Sample requirements and preparation
  • Samples sources include human, animal, natural environments and industrial environments, as well as DNA and PCR products
  • DNA amount: ≥ 500 ng, DNA concentration: ≥ 10 ng/µl, DNA volume: ≥ 50 μl
  • OD260/280 = 1.8~2.0 without degradation or RNA contamination
  • Illumina Nextera XT indexing protocol
Sequencing
  • Illumina NextSeq500 platform
  • Paired-end 150 Bp
  • More than 80% of bases with a ≥Q30 quality score
  • 05M to 1M reads per sample

 

Bioinformatics Analysis

Basic Analysis
  • Quality filtration of data
  • QIIME Analysis
  • Pie charts for each taxonomic class
  • Heat map
  • Abundance and identification of microbial community
  • Taxa identification
  • Rarefaction curve and alpha diversity
  • Comparative analysis
  • Beta diversity (PCoA Plot) (if more than 3 samples)
  • Krona plot
Advanced Analysis
  • Basic+
  • NCBI submission (SRA)
  • Phylogenic analysis
  • Functional analysis of 16S
  • KEGG pathway distribution
  • COG distribution
  • Publication support
Turn-around Time:

6 weeks to deliver the raw data plus 4-5 weeks for analysis depends on the data volume and aim of the study.

Data Storage:

6 months

Metagenomic Shotgun Sequencing

Shotgun metagenomic sequencing allows the taxonomic and biological functional characterization of polymicrobial communities in a cost-effective and time-efficient manner. We at Bioserve consistently deliver high-quality data using Illumina NextSeq platform with flexibility and bioinformatics analysis based on customers’ requirements. The Shotgun method enables microbiologists to evaluate bacterial diversity and detect the abundance of microbes in various environments. Shotgun metagenomics also provides a means to study unculturable microorganisms that are otherwise difficult or impossible to analyze.

Microorganisms are present everywhere in nature. Studies reveal that microbes are critical to these environments and play an essential part in the ecosystem. It arouses an attention for the interpretation of taxonomic composition of a complex microbial community. Shotgun sequencing has been proven as a fast sequencing strategy that uses random fragments. Metagenomic shotgun sequencing is a rapid and powerful tool for obtaining all genetic information in all organisms within a microbial community.

Metagenomic shotgun sequencing targets the entirety of the microbial genetic information contained in an environmental sample. The obtained community taxonomic profile can be further associated with the functional profile of known and unknown organism lineages. The complete sequences of protein-coding genes and full operons in the sequenced genomes can offer invaluable functional knowledge about the microbial communities inhabiting practical ecosystems under study. Metagenomic shotgun sequencing may provide genetic information on potentially novel biocatalysts or enzymes, genomic linkages between function and phylogeny for uncultured organisms, evolutionary profiles of community function and composition and much more. 16S/18S/ITS Amplicon Sequencing is another alternative for metagenomics studies. But this method only interprets the microbial biodiversity, without insights into function.

Unlike capillary sequencing or PCR-based approaches, next-generation sequencing (NGS) allows researchers to sequence thousands of organisms in parallel. With the ability to combine many samples in a single sequencing run and obtain high sequence coverage per sample, NGS-based metagenomic sequencing can detect very low abundance members of the microbial community that may be missed or is too expensive to identify using other methods.

Workflow

Service Specification

Sample requirements and preparation
  • Samples sources including human/animal faeces, natural environments and industrial environments, as well as DNA..
  • DNA amount: ≥ 1000 ng, DNA concentration: ≥ 10 ng/µl, DNA volume: ≥ 50 μl
  • OD260/280 = 1.8~2.0 without degradation or RNA contamination.
  • Illumina TruSeq protocol
Sequencing
  • Illumina NextSeq500,Ion S5 Platform
  • Paired-end 150 Bp
  • More than 80% of bases with a ≥Q30 quality score
  • 2 Gb data per sample

 

Bioinformatics Analysis

Basic Analysis
  • Quality control
  • Marker gene analysis
  • Assembly
  • Compartative genomics
  • Gene prediction
  • Functional annotation
Advanced analysis
  • Basic+
  • Novel genes and functional prediction
  • Biomarker detection
  • Publication support
Turn-around Time:

6 weeks to deliver the raw data plus 4-5 weeks for analysis depends on the data volume and aim of the study.

Data Storage:

6 months

Metatranscriptomics is the culture-independent profiling (including protein-coding and non-coding DNA) of microbial community-wide gene expression. It can monitor RNA-based regulation and expressed biological signatures of complex bacterial communities in a given sample at a given moment and under specific conditions. It elucidates three aspects of a microbial community, including gene activity diversity, gene expression abundance, and differential gene expression analysis. The gene expression analysis can tell us which genes exhibit the highest change in expression levels in different conditions potentially to identify biomarkers and expression signatures.

While metagenomics shows us the microbial species present in the community and their genomic potentials, metatranscriptomics presents the function and activity of the complete set of transcripts, as well as community structure. Metatranscriptomics offers a more informative perspective compared with metagenomics in revealing active biochemical functions, which has become a focus for applications in the environmental, medical, and energy fields as well as the field of drug discovery.

Advantages of Metatranscriptomic Sequencing include:

  • A culture-free method to reveal the true extent of microbial diversity
  • Permitting function-based activity screens
  • More targeted than shotgun random sequencing
  • Cost-efficient and time-effective

Suitable for a wide range of applications, including basic research, ecological applications, clinical applications, industrial applications, and so on.

 
Workflow

Service Specification

Sample requirements and preparation
  • Samples sources including environmental and clinical samples, RNA and cDNA samples
  • The recommended total RNA amount for submission is 6 µg or more with a concentration of greater than 50 ng/µl.
  • The general workflow consists of sample quality control, RNA isolation, purification and qualification; library construction (including fragmentation, end repair, 5’ adaptor ligation, random primer tags and RT-PCR), and library qualification control.
Sequencing
  • Illumina NextSeq Platform
  • Paired-end 150 Bp
  • More than 80% of bases with a ≥Q30 quality score
  • 5 Gb data per sample

 

Bioinformatics Analysis

Basic Analysis
  • Quality control
  • Removing rRNA contamination
  • Annotation
  • DESeq2/EdgeR analysis
  • Taxonomic profiling
Advanced Analysis
  • Basic +
  • PCA, dendrograms and heatmap plots
  • Publication support
Turn-around Time:

6 weeks to deliver the raw data plus 4-5 weeks for analysis depends on the data volume and aim of the study.

Data Storage:

6 months

  • Bacterial Transcriptome Sequencing
  • LncRNA Sequencing
  • mRNA Sequencing
  • small RNA / mi RNA Sequencing
Bacterial Transcriptome 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.

 
Workflow:
 

Sample Requirements:
  • RNA amount: Total RNA≥3 ug (without degradation or DNA contamination)
  • RNA purity: OD260/280 = 1.8~2.2; OD260/230 ≥1.8
  • RNA quality: 28S:18S≥1.5,RIN≥7
Sequencing:

Illumina Nextseq 500,  150 PE, 20M per sample

Data Analysis

Basic Analysis
  • Quality filtration of reads,
  • Read mapping (de novo/ reference based)
  • Summary statistics,
  • KEGG-pathway analysis,
  • Gene ontology analysis,
  • Differential gene expression analysis(if more than one sample),
  • HeatMap, Volcano, MDS/PCA plots
Advanced Analysis
  • Basic+
  • circos, treemap plots
  • Publication support
Turn-around Time:

4 weeks to deliver the raw data plus 4-5 weeks for analysis depends on the data volume and aim of the study.

Data Storage:

6 months

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.

 
Workflow

Service Specification

Sample requirements and preparation
  • Sample type: Total RNA without degradation or DNA contamination
  • Starting amount of Total RNA >2 uG
  • Sample purity: OD260/280 = 1.8~2.2
  • Sample concentration ≥ 200 ng/µl
  • All RNA samples are validated for purity and quantity
Sequencing
  • Illumina NextSeq 500
  • 150 PE sequencing
  • 60M reads per sample.
  • More than 80% of bases with a ≥Q30 quality score

 

Bioinformatics Analysis

Basic Analysis
  • Quality filtration of reads,
  • Identification of Known LncRNAs
  • Expression profiling of LncRNA,
  • Differential LncRNA analysis (if more than one sample),
Advanced Analysis
  • Basic+
  • Novel LncRNA prediction
  • LncRNA effect prediction on the expression
  • Publication support
Turn-around Time:

6 weeks to deliver the raw data plus 4-5 weeks for analysis depends on the data volume and aim of the study.

Data Storage:

6 months

 

 

 

 

mRNA Sequencing

Compared with microarray analysis for known transcripts, mRNA sequencing provides a more precise and complete snapshot of the transcriptome and enables the identification of novel transcripts, alternative splicing, and gene fusion events. RNA-Seq also provides an alternative and affordable approach for gene expression quantification and differential gene expression analysis among groups of samples. It is widely used in disease research, drug response research, pharmacokinetics, and personalized healthcare research.

Bioserve offers complete solutions for transcriptome sequencing and RNA-Seq quantification using a state-of-the-art Illumina NextSeq platform with paired-end 75bp (PE 75) sequencing strategy. Our experienced bioinformaticians work closely with customers to provide standard and customized data analysis and publication-ready results for species with and without a reference genome. Bioserve has experience with >1,000 samples successfully sequenced in over 200 completed projects and articles published on transcriptome sequencing results across different species.

 
Workflow

Service Specification

Sample requirements and preparation
  • Sample type: Total RNA / mRNA without degradation or DNA contamination
  • Starting amount of Total RNA >1 uG
  • Sample purity: OD260/280 = 1.8~2.2
  • Sample concentration ≥ 200 ng/µl
  • All RNA samples are validated for purity and quantity
  • RNA must be validated using an assay such as a BioAnalyzer RNA chip. RIN value ≥ 7.0
  • Animal Tissue, Plant Tissue, cell, blood, and blood products are acceptable.
Sequencing
  • Illumina NextSeq 500
  • 75 PE sequencing
  • 30M reads per sample.
  • More than 80% of bases with a ≥Q30 quality score

 

Bioinformatics Analysis

Basic Analysis
  • Quality filtration of reads,
  • Read mapping (de novo/ reference based)
  • Summary statistics,
  • KEGG-pathway analysis,
  • Gene ontology analysis,
  • Differential gene expression analysis(if more than one sample),
  • HeatMap, Volcano, MDS/PCA plots
Advanced Analysis
  • Basic+
  • Fusion transcript analysis
  • Circos, treemap plots
  • Data modeling and building prediction models
Turn-around Time:

6 weeks to deliver the raw data plus 4-5 weeks for analysis depends on the data volume and aim of the study.

Data Storage:

6 months

small RNA / mi RNA Sequencing

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.

 
Workflow

Service Specification

Sample requirements and preparation
  • Sample type: Total RNA without degradation or DNA contamination
  • Starting amount of Total RNA >5 uG
  • Sample purity: OD260/280 = 1.8~2.2
  • Sample concentration ≥ 200 ng/µl
  • Gel-based miRNA selection
Sequencing
  • Illumina NextSeq 500
  • 1X75 SE
  • 20M SE reads per sample
  • More than 90% of bases with a ≥Q30 quality score

Bioinformatics Analysis

Basic Analysis
  • Quality filtration of reads
  • Identification of known miRNAs
  • Expression profiling of miRNA
  • Differential miRNA analysis (if more than one sample),
  • Potential candidate precursor miRNAs and mature miRNA  identified using a reference genome
  • Secondary stem-loop structures of the candidate (novel) miRNA precursor sequence
Advanced Analysis
  • Basic+
  • miRNA target analysis
  • miRNA target GO and KEGG analysis
  • miRNA and target expression network analysis
  • Novel small RNA prediction
  • Target gene analysis of differentially expressed small RNA (GO enrichment and KEGG enrichment)
Turn-around Time:

6 weeks to deliver the raw data plus 4-5 weeks for analysis depends on the data volume and aim of the study.

Data Storage:

6 months