RNA Sequencing Services
microRNA / Small RNA Digital Gene Expression (mRNA) Sequencing Service Sequencing Service
Next-gen sequencing is a new method and powerful tool The DGE service offered by LC Sciences makes use of used to identify and quantitatively decode the entire RNA Sequencing technology to “count” each individual population of small RNAs in your total RNA sample. LC transcript, offering distinct advantages over semi- Sciences provides a comprehensive sequencing service quantitative array-based gene-expression analysis utilizing Illumina’s high-throughput sequencing systems which rely on signal intensities. As an technology which enables comprehensive, highly alternative to expression arrays, DGE provides a cost sensitive and specific discovery and profiling of all forms effective basic differential expression analysis of known of small RNAs in your sample. genes via the latest next-gen sequencing technology.
Single-Cell RNA-Seq Service Poly(A) RNA-Seq / Total RNA-Seq Sequencing Services Single-cell RNA-Seq can be used to examine the expression of individual cells and provides a higher In addition to expression quantification, RNA-Seq resolution of cellular differences as compared to technology can be used to determine the structure of traditional RNA-Seq. Single-cell RNA-Seq enables us to genes, their splicing patterns or other post understand the function of an individual cell in the transcriptional modifications, and to detect rare and context of its microenvironment. Start with as little as novel transcripts. Total RNA-Seq enables the sequencing 10 pg-10 ng of total RNA. of both poly(A) and non-poly(A) RNAs, such as long non -coding RNA (lncRNA), via a ribosomal depletion library preparation strategy. LC Sciences provides a turnkey solution for transcriptome sequencing featuring the latest in RNA-Seq technology.
www.lcsciences.com 1-888-528-8818
LC Sciences Comprehensive Services
Turnkey Solution These comprehensive services are designed to be one-stop and produce the results needed to quickly advance your biological and biomedical research. For all types of RNA sequencing, our “Total RNA to Data” services include: sample prep/QC, library preparation, RNA sequencing, bioinformatics analysis, and high-level customer support.
Expert Services LC Sciences has been providing RNA discovery, profiling and related bioinformatics services since 2005 and our experts have examined thousands of varied RNA datasets, giving us unique insight into RNA research. We don’t merely deliver data; we will handle all the advanced bioinformatics analysis and help you find answers to the questions you’re asking.
Library Deep Advanced Preparation Sequencing Data Analysis
Key Advantages of RNA Sequencing RNA Sequencing provides a more comprehensive view of the transcriptome. RNA Sequencing enables us to sequence and profile all species of transcripts in your total RNA samples - from mRNA to non-coding RNAs to small RNAs.
RNA Sequencing is not necessarily dependent on any prior sequence knowledge. There is no need for the design of probes that must be based on prior sequence or secondary structure information. Therefore, transcriptome profiling in any species is possible, making this method particularly attractive for non-model species. Additionally, RNA Sequencing data can be used to build de novo gene models.
RNA Sequencing has increased dynamic range and sensitivity. RNA Sequencing enables you to achieve “digital” transcript expression analysis meaning that expression level data are based on each individual transcript that is sequenced and counted. By increasing the sequencing depth, a potentially unlimited dynamic range can be reached making RNA Sequencing an ideal tool for detection rare transcripts.
RNA Sequencing provides information of sequence variation in transcripts. RNA-Seq yields a rich data set that includes information about post transcriptional mutations and their genomic context. Because RNA-Seq data yields information about how exons are connected, it can reveal sequence variations in the transcripts due to alternative splicing events and provide allele-specific or isoform-specific gene expression information. Additionally, RNA-Seq data is useful for gene mapping functions such as describing the length of UTRs and exon boundaries.
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microRNA Sequencing Service
RNA Sequencing provides an excellent tool for discovery of novel microRNAs (miRNAs) as well as genome-wide profiling of small RNA and miRNA expression in various samples. LC Sciences provides a discovery sequencing service utilizing the Illumina high-throughput sequencing technology which enables comprehensive coverage, highly sensitive and specific discovery and profiling of all forms of small RNAs in your sample.
Profile miRNA in any species - there is no need for probes based on prior sequence or secondary structure information. Profiling is transcriptome-wide - investigate all miRNAs and small RNAs, of any size, known and unknown, in your sample. Discover novel small RNAs and detect rare transcripts and transcript variants, such as sequence isoforms and SNPs.
Bioinformatics Package Includes: Raw sequencing data – up to 100M 50bp single-end reads per Alignment, classification, & functional annotation of all mapped lane – either FASTA-A or FASTA-Q files with base-calling quality reads scores Biostatistical analysis – expression analysis, multi-parameter data LC Sciences analysis and quality filtering - processed data is analysis, length distribution, transcript copy number reduced to mappable reads comparisons, etc Custom construction of reference database(s) - miRBase, miRNA target prediction genome, etc and mapping of all quality reads miRNA target GO and KEGG enrichment analysis
microRNA Sequencing Data Flow
Example - pig sample generated ~20M reads. Sequencing reads are mapped to pig mirs in miRBase, mirs of related species and mapped to pig genome.
Group 1 Others Known Pig miRNAs
Group 2 Group 3 Group 4 No Hit Known miRNAs Candidate pig miRNAs Potentially Candidate pig miRNAs genome inconsistent novel miRNAs (713) 664-7087 with miRBase 1-888-528-8818 Fax (713) 664-8181 [email protected]
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Digital Gene Expression (mRNA) Sequencing Service
The incredible power of RNA-Seq was initially applied primarily for complex whole transcriptome applications such as discovery of rare genes, splice junctions and gene fusions or for the study of novel and less well-characterized organisms. However, as the technology is improving and costs are decreasing, RNA sequencing is now suitable to be used for simple expression profiling of known genes as well.
An Alternative to Expression Arrays Achieve digital transcript expression analysis – RNA sequencing technology digitally “counts” each individual transcript, offering distinct advantages over semi-quantitative array-based gene-expression analysis systems which rely on relative signal intensities. Expression profiling is truly genome-wide – there is no need for probes based on prior sequence information. Accurate quantitation of expression levels over 5 orders of magnitude of transcript abundance. Profile gene expression in any species – quantitate differential expression across groups of different biological samples. Provides a cost effective basic differential expression analysis of known genes via the latest next-gen sequencing technology.
Bioinformatics Package Includes: Raw sequencing data – up to 100M 50bp single-end reads per lane – either FASTA- A or FASTA-Q files with base-calling quality scores Data reduced to mappable reads, a list of unique sequences and their copy numbers Genomic mapping of quality sequencing reads Gene expression profiling Test for differential expression at gene and transcript level GO and KEGG annotation and enrichment analysis
Simple report of analysis showing statistical significance of differential expression
Log FPKM FPKM Test_id Gene Locus (fold Test_stat P_value Significant Sample1 Sample2 change)
XLOC_041924 BCLAF1 1:29312111-29312566 7.23 0.30 -3.18 -3.12 0.0018 Yes
XLOC_145478 MOSPD1 X:108883967-108884284 13.84 4.76 -1.07 -3.10 .0019 Yes
XLOC_150271 IRF7 2:294234-294407 11.27 28.54 .93 3.09 .0020 Yes
XLOC_180169 SORBS2 15:44062921-44063290 8.59 0.37 -3.14 -3.08 .0021 Yes
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Digital Gene Expression (mRNA) Sequencing Service Poly(A) RNA-Seq Sequencing Service
LC Sciences provides a turnkey solution for transcriptome sequencing featuring the latest in RNA-Seq technology. Next-gen sequencing is a powerful tool used for transcriptome analysis that can determine the structure of genes, their splicing patterns and other post transcriptional modifications, detect rare and novel transcripts, and quantify their changing expression levels during development and under different disease conditions.
Profile expression in any species - there is no need for probes based on prior sequence information. Compare genome-wide expression in different species or otherwise different biological samples. Detect mutation events such as: novel splicing events, gene fusions, isoforms, SNPs, or other specific coding mutations. Detect rare, or novel transcripts.
Bioinformatics Package Includes: RNA-Seq Reads Raw sequencing data – up to 125M x 2 100bp paired-end reads – either FASTA-A or FASTA-Q files with base-calling quality scores Processed data is reduced to mappable reads
De Novo assembly (if applicable) Align reads to Alignment of RNA-Seq reads to customer specified reference genome genome Identification and construction of splice-junctions Report of known transcripts with annotation and abundance Report of novel transcript with abundance Identification of alternate splicing and report of isoform abundance Test for differential expression at gene level and transcript level Assemble transcripts from spliced alignments GO and KEGG annotation and enrichment analysis More abundant Customer data report - includes a summary of methods and all statistical analysis Less abundant
(713) 664-7087 1-888-528-8818 Fax (713) 664-8181 [email protected] www.lcsciences.com
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Single-Cell RNA-Seq
Single-cell RNA-Seq can be used to examine the expression of individual cells and provides a higher resolution of cellular differences as compared to traditional RNA-Seq. Single-cell RNA-Seq enables us to understand the function of an individual cell in the context of its microenvironment.
Comprehensive Service: Start with as little as 10 pg-10 ng of total RNA Library preparation – poly(A) tail selection method Illumina Hi-Seq 2000 – 2 x 100bp paired-end sequencing run Advanced bioinformatics package
Bioinformatics Package Includes: Illumina base-calling and analysis Raw sequencing data – 50bp single-end reads – either FASTA-A or FASTA-Q files with base-calling quality scores LC Sciences quality filtered data reduced to mappable reads, a list of unique sequences and their copy numbers Genomic mapping of quality sequencing reads Test for differential expression at gene level. GO and KEGG annotation and enrichment analysis. Customer data report – includes a summary of methods and all statistic analysis
Total RNA-Seq (Whole Transcriptome) Sequencing Service
Total RNA-Seq (Whole Transcriptome) sequencing captures a broader range of gene expression changes and enables the detection of novel transcripts in both coding and non-coding RNA species. Ribo-Zero ribosomal RNA reduction chemistry is used in place a poly-A tail selection which efficiently removes ribosomal RNA (rRNA). This process minimizes ribosomal contamination and optimizes the percentage of reads covering RNA species of interest.
Profile expression of any RNA transcript - poly(A) RNA and non-poly(A) RNA. Obtain annotation and expression information on long non-coding RNA within your samples. Sequence non-poly(A) prokaryote transcriptomes. Sequence transcriptomes from degraded RNA from sample types such as formalin-fixed paraffin embedded (FFPE).
Bioinformatics Package Includes: All the analysis provided by the Poly(A) RNA-Seq service, in addition to: Non-coding RNA information – map to genome, alignment, expression analysis and limited annotation Optional circular RNA (circRNA) analysis – although the biological function of most circular RNAs is still unclear, some have shown potential as gene regulators.
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Single-Cell RNA-Seq Degradome Sequencing Service
Degradome sequencing (Degradome-Seq), also referred to as parallel analysis of RNA ends (PARE), incorporates a modified 5′-rapid amplification of cDNA ends (RACE) method with high-throughput next-gen sequencing to provide a comprehensive means of analyzing patterns of RNA degradation. Because miRNAs can cause endonucleolytic cleavage of mRNA by extensive and often perfect complementarity to mRNAs, degradome sequencing has been used to identify miRNA cleavage sites. Degradome sequencing has confirmed many known and revealed novel plant miRNA targets.
In plants, miRNAs tend to cause cleavage of their targets at the position between nucleotides 10 and 11 of the miRNA. Next-Gen sequencing of the 5’ ends of degraded RNA products allows identification of over-represented 5’ ends (miRNA cleavage sites). Matching cleavage sites to known miRNA sequences links miRNAs to their targets.
Bioinformatics Package Includes: Raw sequencing data – up to 100M 50bp single-end reads per lane – either FASTA-A or FASTA-Q files with base-calling quality scores LC Sciences quality filtered data reduced to mappable reads, alignment, classification, & functional annotation of all mapped reads Identification of rRNAs, tRNAs, snoRNAs, polyN and other non-coding RNAs Distribution of degradation fragments on selected region of genome Identification of target mRNAs Statistical summary of mRNA degradation sites Identification of miRNAs from miRBase related to mRNA degradation (results will be shown in a target plot diagram) Customer data report – includes a summary of methods and statistical analysis
Confirmed microRNA targets are presented in the form of target plots (t-plots).
miRNA corresponding to the target cleavage site
Score, Type, P-value
Reads
(713) 664-7087 1-888-528-8818 Fax (713) 664-8181 [email protected]
mRNA www.lcsciences.com
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RNA Sequencing Services
Small/microRNA- Digital Gene Single-Cell Poly(A) Description Degradome-Seq Total RNA-Seq Seq Expression RNA-Seq RNA-Seq
RNA Selection Size Selection – PolyA-tail Selection PolyA-tail Selection – PolyA-tail Selection PolyA-tail Selection rRNA Depletion – method Small RNA – mRNA fragments mRNA – mRNA – mRNA mRNA + lncRNA
Discovery, Discovery, Discovery, miRNA Target ID, Expression, Expression, Data Analysis Expression, Expression, Expression, Degradation Plots Annotation Annotation Annotation Annotation Annotation
Sequencing HiSeq HiSeq HiSeq HiSeq HiSeq HiSeq Instrument
Max# Samples per 10 10 10 5 5 5 Lane
Recommended data size (min 7 million 7 million 7 million 20 million 40 million 40 million reads/sample)
Type of Sample* total RNA total RNA total RNA total RNA total RNA total RNA
Preferred Sample 1 µg 10 µg 3 µg 10ng 4 µg 4 µg Amt
Min Sample Amt 10 ng 5 µg 1 µg 10 pg 2 µg 2 µg Required
Single vs Paired- Single-end Single-end Single-end Paired-end Paired-end Paired-end end Seq Run
Sequencing Run 50 50 50 2 x 100 2 x 100 2 x 100 Length (nts)
Inquire about immuno-precipitated RNAs or other enriched RNAs.
Recent Customer Publications
1. Zhang G et al. (2016) Integrated analysis of mRNA-seq and miRNA-seq in the liver of Pelteobagrus vachelli in response to hypoxia. Sci Rep 6 (1), 22907 [abstract].
2. Han X et al. (2016) Integration of small RNAs, degradome and transcriptome sequencing in hyperaccumulator Sedum alfredii uncovers a complex regulatory network and provides insights into cadmium phytoremediation. Plant Biotech J 14(6), 1470-1483 [abstract].
3. Chen J et al. (2016) Identification of miRNAs and their targets through high-throughput sequencing and degradome analysis in male and female Asparagus officinalis. BMC Plant Biol 16(1), 1 [abstract].
4. Ponce D et al. (2016) Tentacle Transcriptome and Venom Proteome of the Pacific Sea Nettle, Chrysaora fuscescens (Cnidaria: Scyphozoa). Toxins 8(4), 102 [abstract].
5. Sun S et al. (2016) Identification and comparative analysis of the oriental river prawn (Macrobrachium nipponense) microRNA expression profile during hypoxia using a deep sequencing approach. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics 17, 41-47 [abstract].
6. Wang C et al. (2016) A Group of Novel Serum Diagnostic Biomarkers for Multidrug-Resistant Tuberculosis by iTRAQ-2D LC-MS/MS and Solexa sequencing. Intl J of Biol Sci 12(2), 246-256 [abstract].
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www.lcsciences.com 1-888-528-8818