Interpreting missed targets using Tapestri Designer

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Tapestri Designer can fail to find amplicons for 2 broad categories of targets. The first category is amplicon or target proximity related, and prioritization can often rescue so-called ‘must have’ targets. The second category is related to the sequence of the target, and presents a challenge as any potential rescue is only possible by relaxing design criteria (e.g., GC percentage), which may impact amplicon performance . 

 

The Tapestri Designer results include a link to the UCSC Genome Browser for each submitted target that will help in visualizing submitted targets, amplicon inserts, and sequence-related genome characteristics for designs made on hg19 or mm10. 

 

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Clicking this link will take you to the target location with 3 custom tracks loaded:

submitted: (blue): targets that were originally submitted for the design

inserts (green) amplicon insert (= amplicon without primer sequence) 

missed (red) submitted targets that are not covered by any insert.

Primers generally extend ~ 18-35 bp from the insert ends. The amplicon.bed file can be loaded as another custom track and compared to the insert track to visualize primer locations. 

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Amplicon Related 

Tapestri Designer will generate amplicons from ~ 175 -275 bp with primers of ~18 - 35 bp in length. When designing primers, the software will take into consideration GC content of insert and primer, primer melting temperature, primer-primer interactions, secondary structure, primer specificity, and known polymorphism sites. For targets that require more than one amplicon (e.g., > 250bp), tiled, non overlapping amplicons are generated while maintaining the previous considerations. 

White Glove design services for advanced designs, including genome editing, translocations, viral integration, amplicons <175 bp or >275 bp , etc. are available for a design fee. Contact your sales representative or support@missionbio.com for more information.

 

Primer Regions

Regions covered by primers are not considered covered by panels. 

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How to address

Primer regions can sometimes be rescued by adding additional targets in close proximity to the original target(s) to create new amplicons. In the example below, the submitted target is initially missed (middle, red box), as it is located in a primer region (and therefore considered as non-covered). By adding the two new targets, (black boxes), additional amplicons are generated and the middle target is covered.

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Center Gap Regions

Tapestri libraries are recommended to be sequenced with paired-end (PE) 150 sequencing chemistry. For the Read 1 sequence the first ~50 bp are consumed by cell barcode sequence and the next ~20 bp are consumed by primer sequence. This leaves the last ~80 bp as covered. For the Read 2 sequence the first ~20 bp are consumed by the primer sequence leaving the last ~130 bp as covered. Because of this, there may be regions in the middle of amplicons lacking coverage, typically for amplicons longer than 250 bp.

 

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In the UCSC browser you will recognize these types of missed targets as they overlap with the insert-track - illustrating that the target is captured by the amplicon itself and only “missed” when sequencing with paired-end (PE) 150 sequencing chemistry. 


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How to address

Targets can be recovered by sequencing with PE-250 sequencing chemistry. 

Alternatively, center gap regions may be rescued by adding additional targets in close proximity to the original target(s) (see PRIMER REGION paragraph above).

 

Amplicon Tiling

Due to the chemistry of sequencing single cell DNA with limited templates, Tapestri Designer generates tiled amplicons with no overlap. Particularly when targeting whole genes or exons, coverage will be lost due to primer regions and gaps between adjacent amplicons. 

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How to address

Break up longer targets as needed to maintain coverage of ‘must have’ regions. When targeting a whole gene, include hotspots within the gene as additional targets to help Tapestri Designer prioritize these regions.

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Sequence Related 

High GC content

Tapestri Designer will design amplicons in regions with 27-70% GC content. Primers are restricted to 27-62% GC content. Regions outside this GC content are more difficult to amplify by PCR, and the corresponding amplicons are at higher risk to underperform (e.g., low number of reads).  Amplicons generated with GC content 62-70% are shown with a flag in the final design to warn of possible poor performance.

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UCSC Visualization

Enabling the ‘GC Percent’ track can assist in identifying areas of high GC content. DNA sequences can easily be retrieved from UCSC by selecting ‘DNA’ from the ‘View’ menu and used to calculate exact GC percentages for a specific region. View DNA will default to the region currently in view in the browser, or the most recently highlighted region (‘Alt’ + mouse drag Windows, ‘command’ + mouse drag Mac).

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How to address

GC content thresholds help maintain panel uniformity by ensuring amplicons will perform evenly during PCR. White Glove design can attempt to generate amplicons with up to 74% total GC content, but these amplicons may perform poorly and negatively impact downstream analysis. Targets in areas with GC content >74% cannot be rescued. 

Masked Regions

Tapestri Designer cannot generate amplicons in masked regions of the genome. This is any area where the sequence is listed as an N (e.g., centromere regions, telomere regions). Having even a single target with a masked region may cause the entire panel to fail to generate, rather than show as missed coverage. 

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How to address

Masked regions may be rescued using a custom reference genome to supply missing sequence information.

Repetitive Regions

Tapestri Designer may not generate amplicons in highly repetitive regions. In the example below, there is a CAG repeat to the right of the target. The designer cannot generate a unique amplicon for this region.

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UCSC Visualization

Enable the ‘RepeatMasker’ track to help identify repeat regions.

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How to address

If a long repetitive region needs to be covered, White Glove design can assist in generating a panel using PE 250 chemistry to increase read/amplicon length. 

Homologous Regions

Tapestri Designer may not generate amplicons in repeat elements with high homology. LINEs, SINEs, homologous genes, etc may contribute to this.

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UCSC Visualization

Various tracks in the ‘Repeats’ menu can aid in identifying different types of repetitive regions

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How to address

Repetitive regions can be hard masked and uploaded as a custom reference genome. This approach is perhaps most useful for targeting genes duplicated in chrX/Y that cause Tapestri Designer to fail while typically only being present in 2 copies in cells. If designing an experiment with targets repeated in the genome, careful consideration should be taken in terms of panel performance and uniformity, sequencing, and downstream analysis.

 

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