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查看更多产品信息 Ion AmpliSeq™ Exome RDY S5 Kit 4x2 - FAQs (A29855)
24 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.
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DNA outside the region window will not interfere with template preparation or sequencing, but may lead to an overestimation of library concentration when using the Qubit 2.0 Fluorometer for library quantitation.
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Please see here (https://www.thermofisher.com/us/en/home/life-science/sequencing/next-generation-sequencing/certified-service-providers-program.html).
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You can use the Human CEPH Genomic DNA Control from the Ion P1 Controls 200 Kit (Cat. No. 4488985)
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With the normal protocol (assuming the DNA input is correct, i.e., 50-100 ng), you should get about 200-1000 pM without the library amplification. With the library amplification, you would get higher concentration (1-5 nM).
These are average yields, and sometimes this does vary. Keep in mind that the library would work even with lower yields. qPCR is more sensitive than BioAnalyzer for quantitation of yield, and gives a slightly different measurement as qPCR measures ampliplifiable DNA whereas BioAnalyzer just gives the total yield regardless of whether the DNA is good for amplification.
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Here are some suggestions:
- Double check how the DNA was originally quantitated. We recommend using TaqMan RNase P Detection Reagents Kit for quantifying amplifiable DNA.
- If you are still getting low yield using 50-100 ng input, add cycles to the initial amplification. You could try to add 1-3 cycles. It is better to add additional cycles to the target amplification rather than to the 2nd amplification step. Keep in mind that it is important to limit the number of cycles done during the final amplification as you do not want to amplify fragments exponentially as this will introduce bias toward smaller fragments in the amplicon pool.
- Evenness of coverage is affected by bias in “AMP” cycles. Additionally, overamplification will put the sample concentration above the dynamic range of detection for the High Sensitivity BioAnalyzer Chip. It is better to repeat the amplification reaction to generate sufficient product that to overamplify and dilute.
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Yes, up to 3 barcoded exome libraries can be run on an Ion P1 Chip depending on the coverage and depth desired. As the number of libraries on a single chip increases, the average coverage depth decreases.
The recommendation is 2 exomes per chip. We can confidently call germ line mutations at 20 X coverage. You need ˜30 M mapped reads per library, so since the minimum number of reads per chip is 60 million reads, that is 2 exomes per chip.
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- In the Ion Torrent browser, go to the “plan” tab and select “templates”.
- Select “ampliseq.com” and “ampliseq exome”.
- You will be asked to enter your username and password.
- Choose “exome Panel” and select “import selected”.
- Exome template should be created with all appropriates BED and analysis parameter files/json files.
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Make sure that you are using the recommended seals and compression pads (depending on the thermal cycler being used). If you are able to avoid it, we recommend against using Rows A and H (depending on the thermocycler, there can be more evaporation in these outer wells).
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The Ion AmpliSeqLibrary Kit PLUS is a component of the Ion AmpliSeq Exome RDY and Ion AmpliSeq Exome RDY S5 kits, and is not sold separately. It was specifically launched for these kits and has improved formulation components, more robust user variability, higher tolerance of pipetting error, and 1-2% better uniformity.
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All this information can be found here (https://www.ampliseq.com). Each panel has a detailed description, which includes the number of pools, number of amplicons per pool, as well as a “Download Panel Files” link, which will contain the BED files.
The Ion AmpliSeq Exome Panel ships with a smaller, high-confidence exome BED file. For more information about this BED file, see this Technical Note (http://ioncommunity.thermofisher.com/docs/DOC-9357), that describes the Ion AmpliSeq Exome Hi-Q Effective Regions file. This is a BED file in which poor-performing flanking regions have been trimmed away from affected amplicons. To complement this effort, Ion Torrent Variant Calling has also been optimized for the area covered by the Effective Regions File.
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The plates are good for three rounds of target amplification. You can actually cycle the plates with the dried down wells for 3 rounds of cycling, and the dried down wells are still okay, so there is no need to cut the plate.
However, you may want to consider purchasing the Ion AmpliSeq Exome RDY Kit 4 X2 or Ion AmpliSeq Exome RDY S5 Kit 4x2. These kits come with 4 x 96-well plates, each with 2 rows (Rows C and F) filled (4x2). So there are 2 exomes per plate.
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- Ion AmpliSeq Exome RDY Kit 1x8 (Cat. No. A27192) and Ion AmpliSeq Exome RDY S5 Kit 1x8 (Cat. No. A29854) have only one 96-well plate with all 8 rows filled (1x8). Each row of a plate has the 12 primer pools needed for 1 exome. So each plate has 8 exomes (1 exome per row).
- Ion AmpliSeq Exome RDY Kit 4x2 (Cat. No. A27193) and Ion AmpliSeq Exome RDY S5 Kit 4x2 (Cat. No. A29855) come with 4 x 96-well plates, each with 2 rows (Rows C and F) filled (4x2). So there are 2 exomes per plate. The other wells on the plate are empty.
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If barcode balancing is a priority, qPCR is recommended for library quantification. If workflow and speed is a priority, we recommend using the Ion Library Equalizer Kit.
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The Ion AmpliSeq Exome RDY and Ion AmpliSeq Exome RDY S5 kits are not recommended for use with FFPE samples, as the amplicon target sizes (225-275 bp) are larger than we recommend for degraded DNA input. Increasing the input amount will not help, as the issue will be that some or many of the amplicon targets may not be amplifiable due to the degraded (fragmented) nature of the FFPE-derived DNA. This could result in amplicon drop out and incomplete coverage of the intended targets. Further, there could be issues with reproducibility across samples of differing levels of degradation. For example, some samples may produce sufficient results, while others may completely fail or produce sub-par results.
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Due to the number of amplicons and coverage needed, the Ion AmpliSeq Exome RDY panels have only been validated on the Ion Proton Sequencer, Ion S5 and Ion S5 XL Systems.
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The exome RDY primers are dried down into ready-to-use format. There are ˜294,000 primer pairs across 12 primer pools.
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The amplicon size ranges from 225-275 bp, the average insert size is ˜202 bp.
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The Ion AmpliSeq Exome RDY panels cover >97% of CCDS (with 5 bp padding around exons), >19,000 coding genes, >198,000 coding exons, no UTRs, miRNAs, or ncRNAs.
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Ion AmpliSeq Exome RDY Kits contain primers, library reagents, and chips for the rapid preparation and running of 8 exome libraries.
- Ion AmpliSeq Exome RDY Kit 1x8 (Cat. No. A27192) contains the Ion AmpliSeq Exome RDY Panel 1x8 (dried down oligo pools/primers in one 96-well plate with all 8 rows filled (1x8), for ultra-high multiplex PCR enrichment of the exonic regions of the genome)
- Ion AmpliSeq Exome RDY Kit 4x2 (Cat. No. A27193) contains the Ion AmpliSeq Exome RDY Panel 4x2 (dried down oligo pools/primers in four 96-well plates, each with 2 rows (C and F) filled (4x2), for ultra-high multiplex PCR enrichment of the exonic regions of the genome)
Note: Both of the above kits include a 4-pack of Ion PI Chip Kit v3 for sequencing on the Ion Proton Sequencer.
- Ion AmpliSeq Exome RDY S5 Kit 1x8 (Cat. No. A29854) and Ion AmpliSeq Exome RDY S5 Kit 4x2 (Cat. No. A29855) contain all of the same reagents as the Ion AmpliSeq Exome RDY Kit 1X8 and the Ion AmpliSeq Exome RDY Kit 4x2 respectively, but instead include the Ion 540 Chip for use with the Ion S5 or Ion S5 XL Systems.
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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.
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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.
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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.
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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.
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