offers high throughput amplicon sequencing services with fast turnaround times, enabling you to sequence dozens to hundreds of PCR amplicons at once from any source. Amplicon sequencing is based on ultra deep sequencing of PCR products for analyzing genetic variations. Each amplicon molecule can be sequenced individually using either the Illumina MiSeq or NextSeq platform, which have the power to detect rare variants with extremely low detection limits. Ultra deep sequencing enables researchers to focus on a single gene or panel of genes of interest without the high costs of whole genome sequencing, and enables the detection of rare variants with precision, making it ideally suited for experiments looking at the hyper-variable regions of antibodies or population samples for SNP detection, as two examples.
PCR amplicons that are longer than 600 bases in length can be prepared for sequencing using the Nextera XT library preparation kit, which will randomly fragment the amplicons to allow sequencing of all sections. Amplicons shorter than 600 bases can have Illumina-compatible libraries generated through a more direct two step PCR approach.
The pricing for this service depends on the choice of instrument for sequencing. In order to run the samples on the MiSeq, a lane rental must be purchased (see NGS Lane Rentals page), whereas amplicon samples can simply be added onto a NextSeq run at a lower cost and likely with a faster turnaround time. However, the NextSeq can only deliver up to 75bp PE reads, whereas the Illumina MiSeq platform can deliver read lengths of up to 600bp. Since each amplicon-seq project is unique, we encourage you to ask our technical support team for a quote for your custom project.
Key features for abm's Amplicon-Seq service:
• ultra deep sequencing provides highly sensitive detection levels
• targeted sequencing of specific genomic region(s) with minimal off-target reads
• sequence a single sample to hundreds simultaneously, from any source.
• pooling of samples is possible.
• supporting applications for diagnostic approaches.
Sequencing results are delivered in industry standard format (FASTQ) with additional bioinformatics analysis available upon request and/or based on specific project needs. All of abm
's sequencing services are performed by a group of specially trained and experienced scientists partaking in a streamlined workflow.
For more information, please contact our technical support team with details of your project requirements at firstname.lastname@example.org
Sample Requirement Guidelines
Our technical support team will be happy to work on confirming the details of your NGS service. Please contact our specialists at email@example.com to get your order confirmation number. Any samples shipped to our facility without an order confirmation number in the attention line will be destroyed and disposed immediately upon receipt.
Sample Submission Form
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.
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 are the final deliverables from the Amplicon Sequencing Services?
Our Amplicon Sequencing services deliver raw data in FASTQ format as standard. Original SFF/BAM can be requested at the time of ordering for free; but this will need to be requested ahead of time to adjust the sequencing machine output.
PLEASE NOTE: If SFF/BAM is requested AFTER the data has been delivered, this will be charged at a cost if $100/sample to obtain the results in SFF/BAM format.