M13 Human, Forward Primer, HPLC - FAQs

Please see the Application Note (http://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.28891.file.dat/expi293-microtiter-plates-co25793.pdf) for tips on optimizing protein yield using the Expi293 Expression System in 96-well microtiter plates.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Please refer to the Application Note (http://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.61153.file.dat/expi293-protein-yield-co25763.pdf for tips on optimizing protein yield using the Expi293 Expression System.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

If there are issues with the predesigned primers ordered via the Primer Designer Tool, please contact Thermo Fisher Technical Support (https://www.thermofisher.com/us/en/home/technical-resources/contact-us.html).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

To find more information about the status of the primers ordered using the Primer Designer Tool, please contact Thermo Fisher Scientific Product Support (https://www.thermofisher.com/us/en/home/technical-resources/contact-us.html) and have your order number available.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

In the Primer Designer Tool, the amplicon coordinates are displayed for each primer pair. At this time, we do not provide the amplicon sequences from our webpage. To obtain the amplicon sequence, we recommend the NCBI website. You can click on View Details within the Primer Designer Tool and there are several hyperlinks that will take you to different locations on the NCBI page. For example, if you click on the Entrez Gene ID hyperlink, it will take you to the Gene ID page. You can scroll down to Genomic regions, transcripts, and products and click on the FASTA option next to "Go to nucleotide" and that will bring up the whole sequence.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

In the Primer Designer Tool, many of the primer pairs span the entire exon, and hence provide some sequence for exon adjacent sequence. The extent of this intronic sequence varies from assay to assay and the extent can be viewed in the genome browser panel. Currently, we only provide primers for exons and are not offering "intron only" amplicon primers.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The primer pairs obtained via the Primer Designer Tool amplify exon regions of genes. In order to have complete coverage, the first and last primer of each exon typically lies in an intron. There may be some instances where primers are designed in an exon region, due to the size of the exon.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

When using the Primer Designer Tool, the suggested distance of the SNP of interest relative to the 3' end of the primer depends upon the BigDye sequencing kit being used, the use of M13 tails, and the polymer type. BigDye Direct and BigDye Terminator v1.1 typically have higher data quality very close to the sequencing primer end, whereas BigDye Terminator v3.1 together with POP‐7 polymer provides optimized data quality after the first 35 to 40 bases. Predesigned primers ordered with the M13 tails will allow the use of the universal M13 forward and M13 reverse primers for sequencing, the sequence immediately after the M13 primer will be the target primer sequence, and so the SNP can be close to the 3' end of the target primer sequence.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Exon counting is not standardized. Exon 1 often means the first exon on the transcript, but could also mean the first exon on the genome regardless of the sense of transcription. Within the Primer Designer Tool, the mouse over pop‐up‐box on the genomic viewer indicates the number of an exon. However, we recommend user confirmation to ensure that the exon number indicated is concordant with the exon counting system the user expects.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Within the Primer Designer Tool, the following search terms can be used:
- Assay ID: e.g., "Hs00326060_CE"
- SNP ID: e.g., "rs12096805"
- Entrez Gene ID: e.g., "6262"
- Gene Symbol: e.g., "JAK2"
- Gene Name: e.g., "Janus kinase 2"
- RefSeq Accession #: e.g., "NM_007294"
- GenBank mRNA: e.g., "CR536579.1"
- Protein ID: e.g,. "CAA66975.1"
- Cosmic ID: e.g., "121007"
The Primer Designer Tool also allows one to enter a sequence and mapping primer pairs will be returned.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Determining the area sequenced by the predesigned primers ordered via the Primer Designer Tool can be done in the following way:
For each amplicon, the amplicon coordinates are displayed. Clicking on "View Primer on Map" will display the location of the amplicons on the genome and in the context of transcripts.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The typical delivery time frames for predesigned primer pairs ordered using the Primer Designer Tool for USA and Canada are as follows:
- Desalted primers: delivery within 3 business days
- HPLC-purified primers: delivery within 8 business days

- The typical delivery time frames for predesigned primer pairs ordered using the Primer Designer Tool for Europe, the Middle East, and Africa are as follows:
- Desalted primers: delivery within 3 business days
- HPLC-purified primers: delivery within 3-5 business days

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

If your AmpliSeq design has been created using hg38 as a reference, then you can create an ad-hoc workflow in Ion Reporter Software for analysis. All analysis and annotations will take into consideration hg38 as a reference. However, at this moment there are no hg38 workflows in Ion Reporte Software. The tools for analysis and annotations for hg19 will still be available.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

At this moment, there are no new versions of the Oncomine panels based on GRCh Build 38. The Oncomine Panels will still be based on hg19.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

At this moment, there are no new versions of the ready to use AmpliSeq panels based on GRCh Build 38. The off-the-shelf panels will still be based on hg19.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

No, you cannot copy amplicons from an hg19 design to an hg38 design (or vice versa). Amplicons from a custom design can only be copied to another custom design associated with the same reference. It is not possible to copy amplicons to a custom design associated with a different reference.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Yes, the variant calling workflow based on hg19 will still be available in Ion Reporter Software. If your design was created using GRCh Build 38, you can also call and annotate variants using Ion Reporter Software.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Yes. The pipelines and tools for using hg19 as reference for design and analysis will still be available.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Yes, the Variant Analysis and Next-Generation Confirmation cloud apps will support all primers that map to GRCh Build 38. You will be informed with an error message if the primer pair does not map to this release.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

After transition to the GRCh Build 38 build, you should use software and analysis pipelines that still support hg19 to analyze your CE data. CE desktop software, such as Variant Reporter and SeqScape software, will continue to support hg19.

You can also perform analysis using the Variant Analysis (VA) or Next-Generation Confirmation (NGC) cloud apps, but you will need to create a reference using an hg19 Genbank file. If the hg19 Genbank file has annotations, VA or NGC will report out variants based on your provided annotations. In NGC, you will also need to provide the NGS variant file (vcf file) which is generated using hg19 as a reference.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

We cannot guarantee that you will get the same primers if you search by the same coordinates you used for hg19 on the hg38 build.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

No, the AmpliSeq assays and panels that were created using hg19 as a reference should be analyzed with the tools and analysis pipelines created for hg19.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

At this moment, we do not offer any conversion tools to convert coordinates between hg19 and GRCG Build 38. There are publicly available tools to convert coordinates, such as UCSC's LiftOver tool which can be accessed here: http://genome.ucsc.edu/cgi-bin/hgLiftOver

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

We strongly recommend that you download our version of the GRCh Build 38 from our website to use for your analysis of Ion sequencing data. This version is the one that is assumed in all of our software applications.

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Our version of GRCh Build 38 only considers the chr22_KI270879v1_alt. This alt chromosome contains gene GSTT1 that was part of chr22 in hg19.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The differences between the GRCh Build 38 reference used by Thermo Fisher Scientific software and the references publicly available from places like UCSC or NCBI are listed below:
- Repeat and SNP locations are soft-masked into lower case letters, while the ambiguous IUPAC bases, duplicated centromeric arrays, and chrY PAR regions are hard masked into 'N's.
- The version used by our software is based on GRCh Build 38.p2 (http://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.28)
- The GRCh Build 38 reference contains chr1-22, chrX, chrY, chrM, and chr22_KI270879v1_alt.
- Contig chr22_KI270879v1_alt is hard masked except for region 269814-279356 (1-based).
- Gene GSTT1 is located at chr22_KI270879v1_alt:270308-278486.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

We are moving to GRCh Build 38 because it is a more accurate representation of the human genome and is the most recent genome version being accepted by the human genome community. Since many of the annotation databases have moved their content to correspond with the GRCh Build 38 coordinates, this change allows us to link to these newer annotations.

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The GRCh Build 38 is essentially different from hg19 in that the hg19 build is a single representation of multiple genomes whereas the GRCh Build 38 provides alternate sequences ("alt_sequences") for some genomic regions for which their variability prevents adequate representation by one single reference. Other differences between the builds include changes in the centromere representation, the mitochondrial genome, updated sequences, and an "Analysis Set" (FASTA format sequences in a package convenient for use by various Next Generation Sequence read alignment pipelines).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Yes, hg38 and GRCh Build 38 are the same version of the human genome. GRCh Build 38 stands for "Genome Reference Consortium Human Reference 38" and is the primary genome assembly in GenBank; hg38 is the ID used for GRCh Build 38 in the context of the UCSC Genome Browser.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.