Small RNA Sequencing

Description

Small RNAs are a type of non-coding RNA (ncRNA) molecule that are less than 200nt in length. They are often involved in gene silencing and post- transcriptional regulation of gene expression. Our sequencing scale and extensive experience ensure high quality, fast small RNA servcies at unbeatable pricing.

Small RNAs are important functional non-coding molecules, including miRNA (micro RNA), siRNA (Small interfering RNA) and piRNA (Piwi-interacting RNA). They function as inducing sequence-specific gene silence, including RNAi (RNA interfere), translation inhibition, heterochromatinization, inducing gene silencing, to regulate kinds of cell physiological process, such as cell growth and development, stress reaction, and transposon silencing. Unique Molecular Identifiers (UMIs) can be utilized to eliminate undesirable PCR duplicates derived from a single molecule. After PCR, molecules sharing a UMI are assumed to be derived from the same input molecule. As such, UMI counts offer superior results to counting reads, leading to more accurate estimates of quantitative small RNA expression[1]. UMI technology is especially beneficial to customers doing research on rare and precious samples or samples containing less RNAs such as exosomes.

Small RNA sequencing uses next-generation sequencing technology to sequence the miRNAs (18-30 nt or 18-40 nucleotide) of particular tissue in certain physiological status. The sequencing data are then further aligned to database to identify and analyze small RNA sequence, and to annotate the reads as rRNA, tRNA, snRNA, snoRNA and miRNA. Furthermore, it could also predict novel miRNA based on the hairpin structure of pre-miRNA, and identify target gene of known miRNA or predicted novel miRNA. By the related analysis of target gene, such as GO (gene Ontology) and pathway network analysis, association between phenotype and differential expression known miRNA could be founded, so as to explain related physiological phenomenon including flowering time, colors of flower, seed development and disease resistant, pathological regulatory mechanism. etc. DNBSEQTM Small RNA sequencing service with optional UMI technology delivers accurate, affordable and high-quality sequencing data to support academic and clinical research applications with various species.

Discover Our Unique Molecular Identifiers (UMIs) Service

Unique Molecular Identifiers (UMIs) can be utilized to eliminate undesirable PCR duplicates derived from a single molecule. After PCR, molecules sharing a UMI are assumed to be derived from the same input molecule. As such, UMI counts offer superior results to counting reads, leading to more accurate estimates of quantitative small RNA expression. UMI technology is especially beneficial to customers undertaking research on rare and precious samples or samples containing less RNAs, such as exosomes.

History of small RNA sequencing

Northern blot: the earliest miRNA detection method, which is normally used to assess the reliability of other detecting method. The shortcoming is low throughput and weaker sensitivity in testing rare miRNA.

Real-time PCR: use multiplexed reverse transcription (RT) primer pools to amplify low-input miRNAs and quantify kinds of miRNA by distinguishing reverse primer and synthesized primer of the second chain. It could improve reaction specificity and yields as well as detecting sensitivity through incorporating all the miRNAs into muti-RT-PCR system.

miRNA microarray: high throughput ways to screen miRNA with high repeatability, and which is also suited to quantification.

RAKE assay: RNA-primed, array-based Klenow enzyme (RAKE) assay, in which miRNAs are hybridized to immobilized DNA probes on a microarray and are used by Klenow enzyme as primers in an extension reaction. This assay is sensitive and specific for miRNAs and is ideally suited for rapid expression profiling of all known miRNAs, and it could offer unique advantages for specificity over northern blots or other microarray-based expression profiling platforms; miRNA profiling: A new high efficiency miRNA detecting method, which could detect total miRNA amount less than milligram without amplification or distinguishing fragment. The method uses the in situ synthesis probe to selectively match the mature miRNA in sequence and length without sequence shifting, also yields high throughput with low inputting amount of sample. The features of above microarray or microchip technique are high throughput as well as quantification and low input, while the disadvantage is the weak stability of data quality. In addition, there is no inclusive view whether more data mean high quality of these high throughputs testing technology.

SQ-PCR: An amplification-based technique for miRNA quantification (SQPCR, small-target quantitative PCR), the specificity of which is derived in part from the mismatch discrimination afforded by T4 DNA ligase. A reverse transcription step, together with the ligation reaction, targets the sequence specificity of the assay to the central and 3′ terminal regions of the miRNAs, which are the critical regions for distinguishing among miRNA family members. The assay consists of a tandem assembly of enzymatic sub-reactions, which are already used extensively in various assays for nucleic acids. With this robust biochemistry, SQPCR is easily adapted to quantitate any sequence and thus well suited for high-throughput sensitive quantitation of both validated and hypothetical miRNAs.

Technology Advantages

Small RNA provides chance for us to entirely research the composition and function of small RNA in special condition, and provides foundation for us to understand miRNA. Small RNA can correlate DGE or RNA-seq or degradome to research the regulatory mechanism of miRNA and its target genes. Also providing detection of virtually all small RNAs, repeat associated small RNAs, and degraded tags of exons and introns and independent of known information, capable of detecting known miRNAs and predicting and detecting novel miRNAs at the same time.

Key Service Details

Reports and output data files are delivered in industry standard file formats: BAM, .xls, .png and FASTQ data.

Sequencing Standards

• Guaranteed ≥85% of bases with quality score of ≥Q30.
• ≥20 million reads per sample is recommended.

Sample Requirements

Regular Samples:

Mass: ≥ 1μg.

Concentration: ≥ 50ng/μl.

Volume: 15 μl.

General Quality Requirements:

RIN ≥ 6.5 (plant).

RIN ≥ 8.0 (human/animal).

(Note, we can process degraded samples at lower quality thresholds. Contact us for more information.)

Please note: for special samples like exosomes, plasma and other low RNA amount samples, we strongly recommend the customer adding 1ul RNAse Inhibitor when extracting RNA by themselves (Recommending Enzymatics/RNAse Inhibitor/Y9240L/20,000 U).

Turnaround Time

• Typical 27 working days from sample QC acceptance to filtered data availability.
• Expedited services are available, contact us for more detail.

FAQ

Q1: What sample can be accepted in BGI?
A1: Total RNA, recycled small RNA, tissue sample, suspension cell lines and so on.

Q2: What is the minimum input of sample for small RNA library construction?
A2: The minimum amount of sample is 100ng of total RNA.

Q3: How to guarantee the success rate of library construction? What are the QC standards?
A3: The precondition is the quality and the amount of the total RNA. If the requirement is satisfied, the success rate can reach to 95%. The main QC standards consist of the size and the concentration of library, and the qualified library means that the insert size is in the theory range and the concentration meets the sequencing demands.

Q4: What cause failure of library construction?
A4: There are various reasons which can be divided into objective reasons and subjective reasons. Objective reasons include the insufficient amount, the bad quality, the low content of small RNA in samples than normal content and so on, in which the sample itself mainly result. For instance, the small RNA concentration of the serum samples is very low, and the initial input amount of some samples is low too, so, these samples mentioned above could lead to failure of library construction. Subjective reasons
include operation mistake, experiment instrument problem, reagent problem and so on, which have nothing to do with samples. So far, the success rate can reach to 95%.

Q5: What is the sequencing strategy for small RNA? How much data will we get?
A5: We usually run SE50 for small RNA using DNBSEQ platform. 20M clean reads per sample is recommended.

Q6: Is it needed that the partners provide reference sequence for small RNA analysis?
A6: Yes, genome sequence is preferable. If there is no genome sequence, genome sequences or EST sequences of related species is recommended. And some other data is needed. The detailed information refers to the small RNA reference template.

Q7: The way of confirmation of differential expression of miRNAs?
A7: Stem-loop quantitative real-time PCR,Quantitative PCR (qPCR),qRT-PCR

Q8: What samples types are good for UMI small RNA sequencing?
A8: UMI small RNA sequencing can be applied to any types of sample. This technology is especially good for exosomes and samples with limited amount of small RNA.

Q9: For samples with low RNA amount, do we need to add RNAse Inhibitor?
A9: Yes, for special samples like exosomes, plasma and other low RNA amount samples, we strongly recommend the customer adding 1ul RNAse Inhibitor when extracting RNA by themselves (Recommending Enzymatics/RNAse Inhibitor/Y9240L/20,000 U).

Q10: What are special sample types?
A10: For special samples types, such as RNA extracted from serum, plasma samples, tissue fluid samples, FFPE samples, exosomes and low input RNA. Please note the information below:
a. The library cost is higher than the standard sample types due to special treatment.
b. Due to the unexpected sample quality and library quality, the sequencing data amount is NOT guaranteed and we will NOT process additional sequencing for that library for free.

Q11: Why the sequencing data amount is NOT guaranteed for special sample types?
A11: Detailed explanation:
When we sell 20 Million reads/samples, we will set the sequencing order with 20*1.5=30Million raw
reads/library. We would expect 20M clean reads/samples for those standard and qualified samples types. But for those special sample types (especially those sample types in which QC report marked with NA), we are unable to estimate how many raw and clean data we can obtain and we are NOT taking responsibility for the final clean data amount. In this case, additional sequencing fee is required if clients require BGI to provide enough data for that sample. Normally, the data output can be fulfilled, unless some specific cases or specific sample types.

Q12: Do we offer sequencing by lane services for small RNA sequencing?
A12: Yes, we can provide sequencing by lane services for small RNA sequencing, please note that, if sequencing by lane, additional balancing-library with 15% of the sequencing lane is required. The fee for the balancing library is already included in the UMI small RNA sequencing-DNBSEQ SE50 sequencing lane price.