Search
Search
查看更多产品信息 Ion AmpliSeq™ RNA Library Kit - FAQs (4482335)
11 个常见问题解答
A DNA fragment library is constructed from whole genomic DNA and is commonly used for whole genome resequencing or de novo sequencing. Briefly, the whole genomic DNA is fragmented or sheared, ligated with Ion-specific adapter sequences, and then size-selected for the library fragments of the desired length.
Amplicon libraries are constructed from PCR-amplified DNA fragments and are used for targeted sequencing (e.g., investigating variants at known genomic locations). There are two types of amplicon libraries, short and long.
A short amplicon library contains DNA fragments (targets) with lengths that are compatible with the Ion template preparation kits without any further shearing or fragmentation during library preparation. Additionally, no size-selection step is required, as the amplicons are already within the desired size range.
A long amplicon library contains DNA fragments (targets) with lengths that are longer than those compatible with the Ion template kits and requires further shearing or fragmentation during library preparation. The library preparation protocol for long amplicons is similar to fragment libraries.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
Please visit the Ion AmpliSeq Designer website (www.ampliseq.com) and log in with your Thermofisher username and password. Select the “Help” tab to review training videos and documentation, including pipeline details, troubleshooting guides, and frequently asked questions.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
For template preparation, Ion AmpliSeq libraries are diluted to 100 pM, and the volume required for template preparation will vary depending on the template preparation kit used. Please see the Ion AmpliSeq Library Preparation User Guide for details regarding library dilutions and input into template preparation.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
Long-term storage of diluted libraries is not recommended and can result in decreased/suboptimal performance due to the adherence of DNA to the tube. In general, it is best to make fresh dilutions from the library stock for template preparation as needed. Libraries diluted for template preparation may be stored in a sealed plate or 0.2 mL PCR tube at 4-8 degrees C for up to 48 hours.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
A properly stored library (amplified or unamplified) should be stable for at least 1 year at -20 degrees C in a non frost-free freezer. We highly recommend storing the concentrated library in low TE, in a low-bind tube, and placing the tube on the freezer shelf (not door) to minimize temperature fluctuations. To further minimize freeze-thaw cycles, it may also be worthwhile to store multiple aliquots of a single library.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The recommended minimum coverage for germline mutation detection is ~30X, while the recommended minimum coverage for somatic mutation detection is ~500X. Since there will be a distribution of coverage across the amplicons in the library, we recommend targeting an average coverage ~5 times the recommended minimum coverage (150x or 2500x, respectively) to ensure >95% of the bases are covered at the minimum value. In some cases, more or less coverage may be required to achieve the desired variant detection levels.
With Torrent Suite Software v 3.6.2, we support both germline and somatic mutation detection on the Ion PGM and Ion Proton Systems. For the Ion PGM System variant detection down to 5% frequency and Ion AmpliSeq 125-175 bp and up to 275 bp designs are supported. For the Ion Proton System variant detection down to 10% frequency and Ion AmpliSeq 125-175 bp designs are supported.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
Ion Ampliseq technology is a simple, fast, and affordable targeted sequencing strategy based on ultrahigh-multiplex PCR. The library preparation is completed in as little as 3.5 hours using 10 ng of DNA or RNA per PCR. DNA or RNA from a variety of sources—including formalin-fixed, paraffin-embedded (FFPE) tissue—can be used as the starting material.
For DNA input library preparation, you will need the Ion Ampliseq Library Kit 2.0, provided in 8, 96, or 384 reaction formats (Cat. Nos. 4475345, 4480441, or 4480442, respectively) in combination with one of the following Ion Ampliseq panels;
Ion Ampliseq DNA Ready-to-Use Panels:
- Ion AmpliSeq Cancer Hotspot Panel v2 (Cat. No. 4475346)
- Ion AmpliSeq Cancer Primer Pool (Cat No. 4471262)
- Ion AmpliSeq Comprehensive Cancer Panel (Cat. No. 4477685)
- Ion AmpliSeq Inherited Disease Panel (Cat. No. 4477686)
Ion Ampliseq Community Panels (ordered on Ampliseq.com):
- Ion AmpliSeq BRCA1 and BRCA2 Panel
- Ion AmpliSeq Colon and Lung Cancer Panel
Ion Ampliseq Custom DNA Panels (designed/ordered on Ampliseq.com)
For DNA input library preparation, we also offer the Ion Ampliseq Exome Kit (Cat. No. 4487084) as a complete, stand-alone kit.
For RNA input library preparation, you will need the Ion Ampliseq RNA Library Kit, provided in 8, 96, or 384 reaction formats (Cat Nos. 4482335, 4482340, or 4482752, respectively), in combination with one of the following Ion Ampliseq panels;
Ion Ampliseq RNA Ready-to-Use Panels;
- Ion AmpliSeq RNA Apoptosis Panel (Cat. No. 4482571)
- Ion AmpliSeq RNA Cancer Panel (Cat. No. 4482572)
Ion Ampliseq Custom DNA/RNA Panels (designed/ordered on Ampliseq.com)
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
If you've reviewed your design and are not satisfied with the results, please click on the “Not happy with this design? Let us help” link to have an Ion AmpliSeq team member contact you about additional options for your design.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The ~70 bp or ~90 bp peak is likely standard or barcoded adapter dimers, respectively. Adapter dimers may form during the adapter ligation step and are usually removed during the size selection process. The adapter dimers will amplify on the Ion Torrent Ion Sphere particles during template preparation and decrease the overall throughput of usable sequencing reads; thus, we highly recommend removing the adapter dimers by performing an additional clean-up step prior to template preparation.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
In addition to input RNA quality and accurate quantification, the clean-up and size selection steps are critical to generating a successful RNA-Seq library.
- Be sure to mix the nucleic acid binding beads well before dispensing, and follow the workflow and incubation times as closely as possible.
- Use fresh ethanol and pre-wet pipette tips prior to transferring ethanol, as the volume is critical for size selection.
- Remove residual ethanol before elution using a small-volume pipette. Do not over-dry or under-dry the beads.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
Ion AmpliSeq technology offers simple and fast library construction for affordable targeted sequencing of specific human genes or genomic regions. Based on ultrahigh-multiplex PCR, Ion AmpliSeq technology requires as little as 10 ng of input DNA to target sets of genes, making sequencing of FFPE samples routine on Ion PGM Systems.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.