Custom panels undergo the following quality checks:
- High GC Amplicon
- Forward Primer-Amplicon Alignments
- Reverse Primer-Amplicon Alignments
- Mispriming
- Primer Homodimers
- Primer Heterodimers
- Insert-Genome Alignments
- Forward Primer-Genome Alignments
- Reverse Primer-Genome Alignments
Post design in-silico quality checks will help users to assess the performance of the primers or amplicons being designed. While the default parameters used to generate the primers prevent issues like primer dimers, primer specificity to genome, and insert specificity these checks can be useful when pre-designed amplicons are merged with the Designer panel.
NOTE: Please note that these are in-silico checks, an amplicon with multiple warnings may be able to perform well and create desired amplification product, while an amplicon with no warnings may not perform well in experimental conditions. These are an estimate and always look for multiple issues with an amplicon before considering dropping it from the panel.
High GC Amplicon
High GC Amplicons are defined as amplicons with a GC% > 62 for the amplicon sequence, i.e. the primer and the insert region together. The amplicon table highlights such amplicons and shows the GC% for each. For example,
To view the list of all amplicons and their GC%, download and review the QC Summary file in the results zip.
NOTE: Older panels computed the insert GC and not amplicon GC, thus if an amplicon from an older panel is used as a pre-designed amplicon in a custom panel its GC content may change.
Primer Amplicon Alignments
These are the number of reported alignments when primers are aligned to all the amplicon sequences in the panel while allowing up to 2 mismatches. This is an important check as it provides information about cross primer interaction and is especially relevant in case of panel merging. Primers should specifically bind to only their own amplicons and not to any other amplicon region. This check is relevant to Designer generated amplicons as the design process does not check for such cross amplicon interactions once the panel is designed. Efforts should be made to resolve such conflicts in the panel.
Forward primer and reverse primer counts are provided separately. Any amplicon primer which reports more than one alignment is shown as a warning in the Amplicons table. The count (n) shown in the table includes the alignment with its own amplicon so there are (n-1) cross amplicon alignments seen for a primer.
Mispriming
Mispriming is defined as an undesired product formed by a pair of primers (either of same amplicon or different amplicons) that do not overlap any other amplicon region. Any primer that overlaps an amplicon region and forms an undesired product with another primer is flagged by the Primer-Amplicon Alignment checks seen above.
Same Amplicon Mispriming
If the forward and reverse primer from the same amplicon form a product which is either shorter or longer due to the primer aligning to a different location then it shows a mispriming warning for that amplicon. For example,
Longer Product: |
Except, if the product matches what is defined in the design summary file then those amplicons are not flagged. For example, the Myeloid amplicons - MYE_v2_ZRSR2_15841002 which is 145bp in length but does not show a Mispriming warning when used as a pre-designed amplicon.
Cross Amplicon Mispriming
If the primer of one amplicon forms a product with the primer of another amplicon then a misprimed warning is added to both the amplicons as seen below:
The check assumes that the misprimed primers do not overlap within any other amplicon region as seen in the example below:
Case 1: Primers mapping to a completely different location to form a product, both AMPL1 and AMPL2 are assigned Mispriming Warning |
Overlap between Mispriming and other warnings
As the mispriming check helps identify the incorrect products formed by alignment of a pair of primers to an incorrect location, it has an overlap with Primer-Amplicon or Primer-Genome Alignment checks which also flags the primers that are mapping to incorrect location. To make these checks exclusive we give preference to Primer-Amplicon or Primer-Genome warning. Two such examples are given below:
- Interaction between 2 amplicons: As can be seen below a primer of the misprimed pair is at its original location while the other primer overlaps the amplicon and vice versa. In this case, the primer which is aligned correctly has no warning but the other primer has the Primer-Amplicon Alignment warning as seen below:
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Case 3: When a primer overlaps the amplicon completely, none of the amplicons are assigned Mispriming Warning rather Primer-Amplicon Alignment warnings are seen True Amplicons: chr2 TAMPL24_F 12531249 12531277 TAMPL24_R 12531491 12531511 chr2 TAMPL29_F 12553069 12553090 TAMPL29_R 12553221 12553253 (i) Forward Primer-Amplicon Alignment warning for TAMPL29 as the primer contained fully within TAMPL24 chr2 TAMPL24_F 12531249 12531277 TAMPL29_F 12531490 12531511 (ii) Reverse Primer-Amplicon Alignment warning for TAMPL24 as the primer contained fully within TAMPL29 chr2 TAMPL24_R 12553069 12553089 TAMPL29_R 12553221 12553253 |
- Interaction between 3 amplicons: The misprimed product formed by primer of two amplicons overlaps a third amplicon with one primer of the misprimed pair within it while the second primer is aligned outside this amplicon. In this case the first primer will raise the Primer-Amplicon Alignment warning and the second primer will contribute to the Primer-Genome Alignment warning(a warning may not be seen as the threshold for Primer-Genome Alignment warning is >10 occurrences).
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Case 4: Misprimed product overlaps a third amplicon and trigger two different checks True Amplicon: chr2 TAMPL23_F 12547869 12547889 TAMPL23_R 12548090 12548119 chr2 TAMPL24_F 12531249 12531277 TAMPL24_R 12531491 12531511 chr2 TAMPL29_F 12553069 12553090 TAMPL29_R 12553221 12553253
Misprimed product triggers Reverse Primer-Amplicon Alignment warning for TAMPL24 and may be a Reverse Primer-Genome Alignment for TAMPL29 chr2 TAMPL24_R 12547969 12548001 TAMPL29_R 12548135 12548167 |
Refer to this FAQ for more details on such overlaps.
Primer Homodimers
Primer homodimers help identify the primers that may form a secondary structure with itself. Tapestri Designer created panels will not have this issue, but some of the older catalog panels like the Myeloid panel, or in case the primers were designed outside of the Tapestri Designer application this issue can be seen. For example:
Primer Heterodimers
Primer heterodimers help identify the secondary structure a primer may form with other primers in the pool. This check is dependent on the panel size with smaller panels having fewer heterodimers. The default cut-off to assign a heterodimer warning to an amplicon is above 10, which works for up to 500-plex panels. For larger panels, this criteria should be relaxed and 15-20 heterodimers are acceptable.
Insert-Genome Alignments
Insert-Genome Alignments are defined as the number of reported alignments when insert sequence is aligned against the reference genome allowing up to 1 mismatch. Any amplicon reporting 2 or more such alignments will be assigned this warning. If a sequence aligns to more than 1 genomic region, it may lead to loss of reads during analysis as the reads mapping to non-target regions are dropped.
It is important to note that Designer generated amplicons do not show this warning. It can be an issue with the amplicons that are created outside of Designer and used as pre-designed amplicons for merging.
NOTE: The count (n) shown in the table includes the alignment with its own region so there are (n-1) non-specific alignments for an insert.
Primer-Genome Alignments
Primer-Genome Alignments shows the count of reported alignments when primers are aligned to genome sequence with a mismatch of up to 2 and provides insights on primer specificity to the genome. Count of forward and reverse primers aligned to the genome are provided separately.
If a primer aligns to more than 10 regions then this warning is seen. This is again a metric that is not relevant for primers designed in the system. Amplicons from older panels or those created outside of Designer but used as pre-designed amplicons on panel merge functionality may raise this warning.
NOTE: The count (n) shown in the table includes the alignment with its own region so there are (n-1) non-specific alignments for a primer.