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.
What coverage does your Exome Sequencing service offer?
Our 4GB service (IE30501) offers 30X, and 8GB service (IE30502) offers 100X coverage. We can also provide a quote for intermediate (30-100X) or higher (>100X) coverage levels.
Can we identify SNPs or count number variants in for human samples?
If the Exome-Seq Analysis is ordered, we can include the SNP and variant counts. You must let us know your preferred reference - hg19 or UCSD. Our default is UCSD.
What enrichment kit is used?
Our standard Exome sequencing uses Nextera Rapid Capture Exome Kit.
At 30X coverage, we can fit up to 12-24 samples on a flow cell, depending on whether they want to go with the Mid Output or the High Output flow cells.
At 100X coverage, we can fit up to 4-12 samples on a flow cell, depending on whether they want to go with the Mid Output or the High Output flow cells.
What is the sequencing chemistry?
Sequencing will be done on Illumina's NextSeq 500 using the Mid or High Output 150 cycle kit, unless otherwise requested.
Our previous WES delivered ~ 51 Mb capture using SureSelectXT Human All Exon V5 but yours is only 37 Mb. Will many parts of the protein coding genome be missed?
There are many groups that have done comparisons between exome kits, and overall, Illumina and Agilent's kits are fairly similar in coverage. These papers can be found with a quick Google search. If Agilent's SureSelect All Exon Human V5 is preferred, we will be happy to do this for you (additional fees may apply).
Does your advertised 30X or 100X coverage represent minimum coverage or average?
The coverage listed in our services represents an average, as it is nearly impossible to guarantee a minimum coverage level at every single base in the exome.
What is the Read length of your exome seq?
The standard read length is 2x75nt. We can do 2x150nt at additional cost.
How long does bioinformatics analysis take?
Analysis takes between 2-5 weeks, depending on the number of samples submitted and the type of analysis requested. Standard exome-seq analysis can usually be completed quicker than most custom services.
What is the difference between the Nextera Rapid Capture Exome Kit and Nextera Rapid Capture Expanded Exome Kit from Illumina?
The Expanded Exome kit covers more of the regions that fall OUTSIDE of coding regions (UTRs, miRNA, etc). If you are interested in the coding regions, then the regular Exome kit will be better as it targets slightly more exons than the Expanded kit.
Can we submit various concentrations/sample for a total 200ng per sample?
As long as the concentration is >20ng/ul per sample at a total of 200ng per sample, that should be fine.
Can exome-seq be done for any other species than human?
Exome sequencing is reliant on a commercially available kit with the species-specific probes to capture the exonic regions. There are currently no available kits at the moment for any species other than human. You may consider whole genome sequencing however.
What exome enrichment technology do you use for 37Mb Exome?
For exome enrichment, our standard service uses Illumina's Nextera Rapid Capture Exome enrichment kit.
What is the recommended base coverage?
Base coverage depends on what is required by your experiment and analysis. We have options for 30X and 100X as standard services, but we can increase the base coverage if needed.
How do I calculate the amount of reads needed for a specific coverage?
Coverage depends not only on the number of reads, but also on the read length as well. For example, at 2 x 75bp and to get approximately 100X coverage, around 25Million reads is required. The calculation is as follows (for 37Mb Exome):
[ (X mil reads) x 2 x 75 ] / 37 Mb = 60X coverage, and then solve for X.
[25 x 2 x 75] / 37 Mb = ~100X coverage