Description of Ultra Low Input & Single Cell RNA Sequencing Service
Offering next-generation sequencing services for ultra low input and single cell samples is a powerful solution for many research applications. Some of these applications are unable to generate enough sample quantity to meet the minimum input requirements needed for standard library preparation kits, such as stem cell research or working with laser captured materials. Using RNA from a large group of cells can sometimes mask out the unique gene expression of a minor cellular subtype within a larger population of cells, which could potentially lead to missing important data. abm’s Ultra Low Input and Single Cell RNA Sequencing service uses Clontech’s SMART-Seq® v4 Ultra™ Low Input RNA Kit designed for the purposes of sequencing mRNA from low input (10 ng/~1000 cells) or single cell (10 pg) samples. This allows us to analyze samples not previously sequenceable, and open new doors in the clinical research of studying rare tumour subgroups which may be more prominent in driving cancer growth and lead to attractive therapeutic targets. Like our standard RNA-Seq services, we can tailor our sequencing services to a wide spectrum of custom needs and provide minimal turnaround time.
Key features for abm's Single Cell (Ultra Low Input) mRNA-Seq service:
- Unparalleled sensitivity: able to prepare NGS libraries from as little as 10pg (1 cell) of total RNA or polyA+ RNA.
- Employs Clontech SMART technology (Switching Mechanism at 5’ End of RNA Template).
- High quality RNA-Seq data: maintain full length gene information, great representation of GC-rich transcripts.
Included in the Ultra Low Input and Single Cell mRNA-Seq service:
- PolyA Enrichment using the SMART technology.
- Library Preparation and sequencing on the Illumina platform.
- Read Alignment and gene expression quantification for human or mouse (Reference Genome Import Fee may apply for other species).
- QC (RSeQC/FastQC).
- 3 months FREE storage of raw data.
Sample Requirement Guidelines
Any samples shipped to our facility without an order confirmation number in the attention line will be destroyed and disposed immediately upon receipt.
- The maximum input volume of cells or RNA is 10µl. Please ensure your sample is concentrated enough to have at least 10pg-10ng in 15ul. If your RNA sample is not limiting, we recommend you send more total RNA or cells. Purified total RNA should be in nuclease-free water. Cells should be washed and resuspended in 10ul of PBS in a 0.2ml RNase-free individual PCR tube or 8-well strip. Ensure all RNA and cell samples are shipped with sufficient dry ice to keep samples frozen until delivery.
- This protocol has been validated to generate cDNA starting from intact cells; it is possible to use this protocol with previously-frozen cells. The cDNA synthesis protocol has been tested with cultured cells. It cannot be used with cells that have undergone fixation.
- Cells should be washed and resuspended in PBS. The presence of media can interfere with the first-strand synthesis. If necessary, test the effect of your media on cDNA synthesis by performing a reaction with control RNA and the estimated amount of media that you expect to accompany your cell(s).
Sample Recommendations for Ultra Low Input & Single Cell RNA Sequencing Service
- Total RNA Extraction -- The sequence complexity and the average length of SMARTer cDNA are noticeably dependent on the quality of starting RNA material. Due to the limited sample size, most traditional RNA isolation methods may not be applicable. There are several commercially available products that enable purification of total RNA preparations from extremely small samples [e.g., Clontech offers the NucleoSpin RNA XS kit (Cat. No. 740902.10) for purification of RNA from ≥100 cells]. When choosing a purification method (kit), ensure that it is appropriate for your sample amount. Input RNA should be free from poly(A) carrier DNA that will interfere with oligo(dT)-primed cDNA synthesis.
- Evaluation of RNA Quality -- After RNA extraction, if your sample size is not limiting, we recommend evaluating total RNA quality using the Agilent RNA 6000 Pico Kit (Cat. No. 5067-1513).
- Cell Culture Media -- When working with cultured cells, it is important to select a cell culture medium that does not inhibit first-strand cDNA synthesis. The protocol in this user manual was validated with cells suspended in cell culture-grade PBS.
Confidentiality of Ultra Low Input & Single Cell RNA Sequencing Results
All customer information is held in strict confidence. All materials and information sent to us and the products produced by us for the order are the property of the customer and will be returned to the customer or discarded in a confidential manner. We only archive customer materials when instructed to.
Please email us at NGS@abmgood.com for assistance with starting your Ultra Low Input & Single Cell RNA-Seq project or any inquiries about our next generation sequencing services. We offer discounts on bulk sample sequencing please inquire for more details. ABM also provides an RNA purification service from original tissue samples for $50/sample. The minimum amount of RNA needed depends on the specific RNA preparation method pertinent for each project. A sample preparation guide for RNA Sequencing with ABM can be downloaded here.
Our goal is always to deliver high-quality useful data to aid you in your research. We perform extensive maintenance and quality assurance on our equipment and strict quality control of samples throughout the sequencing process.However, next-generation sequencing by nature does not always deliver equally robust results due to a variety of factors, such as the variability inherent in the library preparation process, the biochemistry that takes place during sequencing and the quality of samples that we receive. We will make every effort to deliver the number of reads that are listed, although it is not possible to guarantee that those targets will always be achieved. The actual number of reads passing filter may vary by up to 10% from the listed target. Studies by various institutes have shown that there is minimal difference in coverage for 10% variation in reads past 5-10 million total reads.
Is your company certified as a service provider for Illumina sequencing?
Yes, we are a certified Illumina service provider.
What kind of analysis software do you use for sequence analysis?
The Illumina sequencers come with the integrated software that can perform the regular bioinformatics analysis functions. In addition, we have extensive experience with other open access software such as Galaxy, Adnuril, BioBike, etc. In other words, we are willing to perform additional bioinformatics analysis with different software and tools to satisfy a variety of demands from our customers.
What kind of QC methods do you adopt for the customer's sample(s)?
We will perform QC on your Total RNA samples prior to sequencing them, using the Agilent Bioanalyzer (industry standard) to determine the RNA Integrity Number (RIN). If the RIN is lower than 8, we consider these samples as having failed QC. If the samples do not pass QC, we will ask you to send us new samples. If we do not receive new samples, thereby cancelling the sequencing order, there will be a QC fee of $50/sample. This fee will be waived if you choose to continue with the sequencing order. A strict positive and negative control will be applied to this QC process.
The library QC will also be performed using the Agilent Bioanalyzer to determine library size and purity. Also, prior to loading the libraries on the sequencer, we perform qPCR quantification. The cost for this is included in the sequencing service.
For the QC of the final data from our sequencing service, the integrated software will generally do the job and provide adequate QC information, however, we can also provide additional QC data such as FASTQC upon request.
The data we output will pass our Q30 filter, which means that the error rate in base calls is less than 1 in 1000, or 0.1%. We will get a percentage at the end of the run which states the percentage of bases that have a Q score > 30, and this percentage is usually ≥85%.
What is the lead time for your WGS, WES, and RNA-Seq services?
Our sequencing lead times depend on workload, but are typically 2-4 weeks for sequencing data + 2-3 weeks for standard analysis. Please inquire for lead times for more detailed analysis.
Can you recommend any preferred RNA/DNA isolation kits to provide the best quality samples for these services?
For miRNA sequencing, you may isolate your RNA sample with a mirVana™ miRNA Isolation Kit,
mirVana™ PARIS™ Kit or PureLink® miRNA Isolation kit to enable adequate enrichment of your sample. If preferred, it should be okay to use Qiagen's kit, as long as the RNA Integrity Number (RIN) of the sample is above 8.0 to make sure there is no significant degradation. (Samples with a RIN lower than 8.0 may contain smaller degraded RNA fragments that can be sequenced in addition to the miRNAs). We prefer receiving Total RNA sample however so we can perform the bioanalyzer QC to check the RNA quality before beginning library construction. There will be no extra-charges if you submit Total RNA instead of isolated miRNA.
For miRNA sequencing we require 200ng-2ug of total RNA in 10ul of nuclease free water, as quantified with a fluorometric method. Lower amounts might result in inefficient ligation and low yield. If you are supplying purified miRNA, please submit a minimum of 50-100 ng of purified small RNA in 10ul is required. Purified small RNAs must be in nuclease free water or 10 mM Tris-HCI, pH 8.5.
For other services, there are generally no preferred DNA/RNA isolation kits as long as minimum requirements for QC are met.
Do I need any special software to view the data report files?
You will first need to unzip the fastq.gz files with winrar to view with vim, nano, gedit, Notepad plus plus. Nextgen workbench generally can view the data immediately without unzipping.
The RNASeq_sample_cufflinks_output.tar.gz will also need to be unzipped to see the text files by vim, nano, gedit, or Notepad plus plus. The unzipped data an also be imported as text into the data tab of Excel 2007. Other versions may differ.
To analyze further, Excel can sort, filter, find, match, etc and has many functions for conditional coloring, graphing, etc.
For the data, why are there no "ALT", "POS" and "ID" in your CSV file?
CSV files are meant to be used in conjunction with the raw fastq/bam files by certain commercial bioinformatics software. Many have a proprietary algorithms that takes the ALT POS and ID from the Fastq and indexes them for its own use. They are not meant to be used alone.
Would you let me know the description for the following: Genotype Sample_0, Coverage Sample_0, Coverage By Allele Sample_0, Variant Quality Sample_0, and Genotype Quality Sample_0?
Genotype (Sample Name) = nucleotide. Coverage (Sample Name) = will fail if there are insufficient reads. Coverage by allele (Sample Name) = will fail on one strand (+/-) read if only one side of the pair was sequenced. Variant Quality (Sample Name) = confidence of variant called. Genotype Quality (Sample Name) = confidence of genotype sequenced.
Concerning the column of "CHROM", would you let me know the rules of naming?
type of accession number| actual number| type of sequence| actual curated version used
Does your NGS service including RNA isolation?
We do not provide RNA isolation service by default. However we can provide this service. For more information, please refer to Cat#IR19001.
How many biological replicates do I need for each condition?
To increase confidence and reduce experimental error it is suggested that you submit at least 3 replicates per sample. Note that this is to serve as a guideline only and the final number of replicates and samples is to be determined by the end user based on their final experimental conditions.