Sample Submission Guidelines
Frequently Asked Questions
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Sample delivery
- 1.What starting materials does CD Genomics accept for the services?
- We accept total RNA, genomic DNA, amplicon, cell cultures and pellets, tissues, blood, etc.
- 2.We will have several samples collected in different time points. Should we do sequencing when we collect all samples?
- You may send samples to us in several batches. This will allow us to run QC first to check for the sample quality. If sample does not pass QC, this will give you some time to prepare new samples, and we will do the sequencing when we receive all the samples.
- 3.Can you recommend any preferred RNA/DNA isolation kits to provide the best quality samples for NGS services?
- For RNA-seq, Trizol is more commonly used, you can just use the Chloroform/Phenol method for purification as instructed in the Trizol kit. Another easier method is to use the commercial kit (e.g. Qiagen RNeasy Mini Kit). Moreover, for isolating total RNA from plant samples, we would recommend Qiagen RNeasy Plant Mini Kit.
For miRNA sequencing, you may isolate your RNA sample with a mirVana™ miRNA Isolation Kit or Qiagen's kit. We prefer receiving Total RNA sample however so we can perform the Bioanalyzer QC to check the RNA quality before beginning library construction.
For other services, there are generally no preferred DNA/RNA isolation kits as long as minimum requirements for QC are met.
- For RNA-seq, Trizol is more commonly used, you can just use the Chloroform/Phenol method for purification as instructed in the Trizol kit. Another easier method is to use the commercial kit (e.g. Qiagen RNeasy Mini Kit). Moreover, for isolating total RNA from plant samples, we would recommend Qiagen RNeasy Plant Mini Kit.
- 4.How long does CD Genomics keep my samples for? Do you send the left over samples back to me?
- Due to limited space in the DNA Sequencing facility freezer we only keep your samples for 2 months, after which time they will be discarded without warning. If you would like your remaining samples returned to you please contact us, we will charge shipping/handling fee.
- 5.Will the long-distance transport (e.g. international) affect the sample condition?
- By using proper shipping approaches, the transport will not affect the sample condition. For DNA samples, most DNA are stable in ice for a few days, the dry ice will not require. For RNA samples, RNA is much less stable than DNA, you can send with dry ice, with ethanol precipitate or using RNAstable reagent. For tissue sample, you can send in RNAlater reagents and ship with room temperature.
Please seal each vial tube to absolutely prevent leakage and breakage and evaporation, and a secondary container is recommended.
- By using proper shipping approaches, the transport will not affect the sample condition. For DNA samples, most DNA are stable in ice for a few days, the dry ice will not require. For RNA samples, RNA is much less stable than DNA, you can send with dry ice, with ethanol precipitate or using RNAstable reagent. For tissue sample, you can send in RNAlater reagents and ship with room temperature.
- 6.Do you accept FFPE samples for WES and RNA-seq?
- Yes. For WES, we can use degraded DNA. For degraded FFPE RNA, we use Ribo-Zero technology to deplete rRNA, as the normal protocol will not work due to loss of the poly-A tails.
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Technology platform
- 1.Which sequencing platforms do you have?
- We have the Illumina sequencing platforms and PacBio SMRT Sequel sequencing platform, and we are setting up the Oxford Nanopore platform.
- 2.How do you guarantee the sequencing quality?
- We will provide customers with the QC report in every major steps, i.e. QC of the initial input sample, library preparation QC and raw data QC to detect the sequencing quality. Nevertheless, be ensure to do your own quality control before you ship the samples in order to avoid delays or other pitfalls in processing.
To monitor sequencing quality control, the PhiX Control will be used. Illumina PhiX Control can be used as a positive control in the clustering process. If a problem occurs in sample preparation, PhiX still generates clusters. These clusters help to discern whether a lack of clusters is due to sample preparation failure or a failure in the cluster generation process. And we will repeat the sequencing step if the failure is due to the sequencing run.
- We will provide customers with the QC report in every major steps, i.e. QC of the initial input sample, library preparation QC and raw data QC to detect the sequencing quality. Nevertheless, be ensure to do your own quality control before you ship the samples in order to avoid delays or other pitfalls in processing.
- 3.What kind of methods do you use for QC?
- Typically, we would perform sample QC using the Qubit, NanoDrop and Agarose Gel to detect the quantity of sample and using Agilent Bioanalyzer (industry standard) to determine the RNA Integrity Number (RIN).
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 generated 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.
- Typically, we would perform sample QC using the Qubit, NanoDrop and Agarose Gel to detect the quantity of sample and using Agilent Bioanalyzer (industry standard) to determine the RNA Integrity Number (RIN).
- 4.How many biological replicates do I need for each condition?
- To increase confidence and reduce experimental error, it is strongly suggested that you submit at least 3 replicates for each treatment/group. Please 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. We do not make technical replicate libraries from the same sample, as next-generation sequencing technology itself is highly reproducible.
- 5.Do you pool samples/libraries in one sequencing lane?
- Yes, we can multiplex your samples and reduce the average sequencing cost for each sample.
- 6.If we don’t have enough input material as you suggest, can you still proceed?
- We can apply the low input DNA/RNA library preparation strategy. Alternatively, we can also offer an option for DNA/RNA amplification, followed by library preparation.
- 7.Do you accept self-prepared libraries?
- Yes, and we will do Bioanalyzer for qualification before loading into the sequencer. We require at least a 10 nM pooled library.
- 8.What technology do you use for SNP genotyping?
- We provide SNP genotyping services in both whole genome scale and in targeted loci.
For whole genome SNP genotyping, we provide whole genome sequencing by using next generation sequencing technology, and we also provide SNP microarray by using commercial arrays.
For targeted loci genotyping, we can use multiple technologies including MassARRAY SNP genotyping, SNaPshot and KASP technology by using next generation sequencing. We can provide free consulting to design properly approaches for your requested project.
- We provide SNP genotyping services in both whole genome scale and in targeted loci.
- 9.What advantages does RNA-Seq have over using Gene Expression Profiling Microarray?
- Compared to microarray-based methods, RNA-seq provides far higher coverage and greater resolution of the dynamic nature of the transcriptome, which is more cost-effective. We have the cutting-edge Illumina platform, with the PE150 sequencing strategy (an average of 6Gb data per sample) and strong data analysis ability, to support your research.
- 10.When is it necessary to eliminate rRNA from my samples before sequencing, and what’s the advantage by doing that?
- In total RNA, rRNA accounts for more than 90%. Performing RNA-Seq without Poly (A) enrichment or rRNA depletion will result in most reads coming from ribosomal RNA Thus, Poly(A) enrichment or rRNA depletion is necessary for any sequencing platform.
- 11.Do we need to perform miRNA isolation for miRNA sequencing?
- There is no need to purify miRNA since the NEBNext Small RNA Library Prep kit uses total RNA as the starting material. This kit uses special 3' and 5' adapters that will ligate to miRNAs specifically and not to rRNA or other transcripts. DO NOT use the column kit that may filter out the small RNA.
- 12.What if the sequencing fails?
- Firstly, we do QC in every major step to ensure the success of the project. But the sequencing can sometimes fail for many reasons, including failures on our part, e.g. instrument malfunction, technician error and failures on customer, e.g. impure sample, misdesigned experiment. We will, to the best of our ability, assess whether a failed analysis is due to a failure on our part, and we may in such cases repeat an analysis at our cost. We will not be held liable for sequencing failures arising due to errors or problems in the Client’s laboratory.
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Analysis report
- 1.How do I get the raw data?
- Raw Data and analyzed results can be transferred via secure, password protected FTP.
- 2.What kind of special software is needed 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++. Nextgen workbench generally can view the data immediately without unzipping. The unzipped data can be imported as text into the data tab of Excel.
- 3.What kind of data do you deliver?
- The raw NGS data we delivered will be clean fastq files. And we also send a report for your project. If you select our bioinformatics analysis service, additional files such as bam files, vcf files and more (depending on the analysis), will be delivered.
- 4.How long will my project data be stored?
- We store your data for 3 months after data generation without additional charge. You can ask us to keep it for longer (at cost).
- 5.Should I provide a reference genome for data analysis?
- Besides resequencing many applications such as transcriptome sequencing rely on a high quality reference genome as basis for the deeper analysis. The full reference sequence ensures where to count, while the full reference annotation ensures what to count. Additional annotations of a reference genome allows additional deeper analysis such as pathway analysis, alternative transcript detection.
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Schedule
- 1.What is the turnaround time for your NGS services?
- The turnaround time will be dependent on the number of samples to be tested, the type of run, the nuclear acid extraction and data analysis requirement and other factor. Regularly, the timeframe of library construction and sequencing is ~ 30 business days. When we set up a project with you we will give you an estimate of how long we expect it to take.
- 2.What's the typical validity period of quotation?
- The validity of quotation is 60 days.
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Privacy security
- 1.Do I own intellectual property of my results? And how you can ensure the security of our data?
- Yes, we are a service provider, and as such, we do not claim any intellectual property. We will keep all the data confidential, and promise to not share the data to anyone else without your permission.
For research purposes only, not intended for clinical diagnosis, treatment, or individual health assessments.
