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Ion Total RNA-Seq Kit v2, 48 reactions - FAQs

Ion Total RNA-Seq Kit v2, 48 reactions - FAQs

查看更多产品信息 Ion Total RNA-Seq Kit v2 - FAQs (4479789, 4475936)

14 个常见问题解答

What is an indicator of a successful RNA library?

Generally, a successful Ion RNA-Seq library will be within the recommended size range for sequencing on the Ion PGM or Proton systems and the sequencing data will have a high (or expected) percentage of on-target reads.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

I am new to RNA-Seq. Where can I find information on how to approach the sequencing data analysis?

Please visit the Transcriptome Sequencing by Ion Torrent Sequencing page (https://www.thermofisher.com/us/en/home/life-science/sequencing/rna-sequencing/transcriptome-sequencing/transcriptome-sequencing-ion-torrent-next-generation-sequencing.html) for information on RNA-Seq data analysis using Torrent Suite Software, or 3rd party software providers.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

Why do I see the sequence GGCCAAGGCG at the beginning of the majority of the reads in my sequencing data analysis?

The GGCCAAGGCG sequence is part of the RNA library adapter sequence. This sequence can be automatically trimmed by the software by selecting the “RNA_Barcode_None” option under the Barcodes menu. The run can be reanalyzed with the correct barcode settings to trim the adapter sequence. In the future, the “RNA_Barcode_None” option should be selected in the run plan or selected on the instrument prior to starting the sequencing run.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

Which Ion platform and chip should I use for whole transcriptome sequencing?

RNA-Seq relies on a random sampling strategy in which most sequencing reads will identify abundant transcripts with an increasing number of reads needed to identify lower abundant transcripts and/or low levels of gene expression. While depletion and purification strategies are available from Thermo Fisher Scientific to remove unwanted abundant transcripts and increase sequencing power, transcript detection sensitivity is a function of mapped reads. Accordingly, the complexity of the genome under investigation should be taken into account when planning an RNA-Seq experiment. Applications that require a small number of reads, such as low multiplexed gene expression, can be performed on the Ion 318 Chip Using the PGM platform whereas, human/mammalian transcriptomes and highy-multiplexed gene expression profiling should be performed on the GeneStudio platforms, using the higher throughput chips. For more information, please see the Transcriptome Sequencing by Ion Torrent Sequencing page (https://www.thermofisher.com/us/en/home/life-science/sequencing/rna-sequencing/transcriptome-sequencing/transcriptome-sequencing-ion-torrent-next-generation-sequencing.html).

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

The Ion Total RNA-Seq Kit v2 utilizes RNase III for RNA fragmentation in the whole transcriptome library preparation workflow. Are there other forms of fragmentation?

Another method is chemical fragmentation (not part of the protocol), which is useful for quantifying exon levels, novel splicing identification, and fusion gene discovery, and provides more uniform coverage for viral and bacterial transcripts. Chemical fragmentation is more randomized than RNase III fragmentation, but can be difficult to optimize. Chemically fragmented RNA fragments require repair with T4 polynucleotide kinase (not included in kit) before proceeding into ligation.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

My final library yield is poor. What are some tips for improving my yield?

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 aliquoting 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.

Why does the Ion Total RNA-Seq Kit v2 (Cat. No. 4475936) specifically require the Ion Xpress RNA-Seq Barcodes 1-16 Kit (Cat. No. 4475485)? Why can't I use the DNA barcodes provided in the Ion Xpress Barcode Adaptors 1-96 Kit (Cat. No. 4474517)?

The Ion Total RNA-Seq Kit v2 workflow uses a different method for adding the barcode sequences to the library than the standard Ion DNA libraries. For standard DNA library construction, the barcoded adapters are directly ligated to the DNA inserts. For Ion Total RNA-Seq libraries, the barcode adapter sequence is added during the final library amplification.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

What is the difference between the current Ion Total RNA-Seq Kit v2 (Cat. No. 4475936) and the original Ion Total RNA-Seq Kit (Cat. No. 4466666)?

The current Ion Total RNA-Seq Kit v2 Kit is compatible with barcoding, uses bead-based size-selection, and has higher fidelity polymerases for reverse transcription (RT) and PCR (SuperScript VILO and Platinum PCR SuperMix High Fidelity).

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

What if I used a mixed RNA sample for input (e.g., viral RNA mixed with mammalian tissue)? Can I use it for constructing an Ion RNA-Seq library targeting the viral RNA?

The mixed library will result in inefficient sequencing, a large proportion will be off-target, non-viral sequence. If possible, it is best to separate the sample before RNA isolation.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

Are there any protocol modifications when using degraded RNA as input into library preparation?

For whole transcriptome library preparation with FFPE samples, the RNA may be degraded enough to omit the fragmentation step and proceed directly to adapter ligation. In these cases, the FFPE-derived RNA will benefit from an additional kinase step prior to adapter ligation and an overnight ligation reaction. We recommend contacting your local Field Applications Scientist if working with degraded samples.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

What are the input requirements for the Ion Total RNA-Seq Kit v2 (Cat. No. 4475936)? Is there a recommended quality for the RNA input?

For whole transcriptome library preparation, best results are obtained with an RNA Integrity Number* (RIN) > 7. Poorer quality RNA can still generate libraries. However, the degraded RNA will be harder to separate out by size or other properties (e.g., rRNA-specific sequences, poly(A) tails), which will result in more off-target sequencing.

Whole Transcriptome (polyA-selected or rRNA-depleted RNA is highly recommended, but total RNA is acceptable)
- PolyA-selected RNA: 1-500 ng
- rRNA-depleted RNA: 10-500 ng
- Total RNA: 1-100 ng

*The RNA Integrity Number (RIN) is obtained by evaluating the RNA sample using the appropriate Agilent RNA Kit for the Agilent 2100 Bioanalyzer instrument.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

What kits should I use to isolate RNA from my sample?

The best isolation kit to use will depend greatly on your application, but, in general, the kits below produce RNA input compatible with the Ion Total RNA-Seq Kit v2 workflow:

Whole Transcriptome (polyA-selected or rRNA-depleted RNA is highly recommended, but total RNA is acceptable)
- Poly A-selected RNA
• Dynabeads mRNA DIRECT Micro Kit (Cat. No. 61021)
• MicroPoly(A)Purist Kit (Cat. No. AM1919)
- rRNA-depleted RNA
• RiboMinus Eukaryote System v2 (Cat. No. A15026)
• Low Input RiboMinus Eukaryote System v2 (Cat. No. A15027)
- Total RNA
• TRI Reagent
• TRizol
• RecoverAll MagMax

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

How does library preparation with the Ion Total RNA-Seq Kit v2 maintain strandedness?

Unique 5´ and 3´ adapters are directionally ligated to RNA prior to reverse transcription, preserving the strand information through the rest of the workflow.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

I am new to making RNA libraries using the Ion Total RNA-Seq Kit v2 (Cat. No. 4475936). What is a good approach?

Please visit the RNA Sequencing applications (https://www.thermofisher.com/us/en/home/life-science/sequencing/rna-sequencing.html) for information regarding sequencing data analysis, including white papers, training videos for transcriptome data analysis, and information about third-party software solutions (i.e., Partek Inc.).

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.