Load Samples

Loading Loom File

Reorder box   Drag and drop this box to reorder the samples. All downstream analysis will maintain this order for visualization.
Include x Uncheck to remove the data from analysis.
Label Give the sample a biologically meaningful name.
Filename Name of the loaded file.
Cells Total cells found by the primary analysis pipeline
Total Reads Total reads from the sequencing run
Reads/Cell Average reads per cell. Only using reads that mapped to cells. Reads with unidentified cell barcodes are excluded from this calculation.
Reads/Amplicon/Cell Average reads per amplicon per cell. Only using reads that mapped to cells. Reads with unidentified cell barcodes are excluded from this calculation.

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Loom Files

Tapestri pipeline outputs the data in loom file format. LOOM is an efficient file format for very large omics datasets, consisting of a main matrix, optional additional layers, a variable number of row and column annotations. We’ve added multiple layers to this file format to accommodate single cell genotype data. Tapestri Insights uses the file format to quickly read and analyze large amounts of single cell genotype data.

Loom files from other pipelines will not work with Tapestri Insights. Make sure you are only working with LOOM files that are generated with the latest Tapestri Pipeline.

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White list

A list of known chromosomal locations with (pathogenic) variants. Variants that match the white list will not be affected by the filtering parameters. Genotype calls for these variants will remain exactly as output by GATK pipeline.

The format of the whitelisted variants follows the BED file format and needs to conform to tab-delimited text file format that defines a specific feature track. It can have any file extension, but BED is the most common.

The example below shows 2 feature tracks. The first track searches for putative variants at the genomic positions from 77210085 to 77210090 on chromosome 10. The second track searches for putative variants at the genomic position 19203444 on chromosome 20.

Note that at positions at which the Tapestri Pipeline detects multi-allelic variants all variants will be listed in Tapestri Insights.

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Cell-Specific Filters

(1) Remove genotype in cell with quality < X

The genotype quality score represents the Phred-scaled confidence that the genotype assignment (GT) is correct, derived from the genotype normalized likelihoods of possible genotypes (PL). Specifically, the quality score is the difference between the PL of the second most likely genotype, and the PL of the most likely genotype. The values of the PLs are normalized so that the most likely PL is always 0, so the quality score ends up being equal to the second smallest PL, unless that PL is greater than 99. In GATK, the value of quality score is capped at 99 because larger values are not more informative, but they take more space in the file. So if the second most likely PL is greater than 99, we still assign a quality score of 99.

Basically the GQ gives you the difference between the likelihoods of the two most likely genotypes. If it is low, you can tell there is not much confidence in the genotype, i.e. there was not enough evidence to confidently choose one genotype over another.

We recommend using a value of > 30.

(2) Remove genotype in cell with read depth < X

The Read Depth per Variant metric is the filtered depth, at the cell level. This gives the number of filtered reads that support each of the reported alleles.

We recommend using a value of < 10.

(3) Remove genotype in cell with alternate allele freq < X

The Alternate Allele Frequency is the unfiltered allele depth, i.e. the number of reads that support each of the reported alleles. All reads at the position (including reads that did not pass the variant caller’s filters) are included in this number, except reads that were considered uninformative. Reads are considered uninformative when they do not provide enough statistical evidence to support one allele over another. Only non-reference genotype calls are included.

We recommend using a value of < 20 %.

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Global Filters

(4) Remove variants genotyped in < X of cell

The Cells with Min % of Genotypes Called metric is the proportion of genotype information available on a per-cell basis. For example, a threshold of 80% retains only cells in which at least 80% of genotype information is available for any given variant.

We recommend using a value > 50 %.

(5) Remove cell with < X of genotypes present

The Genotypes Called in Min % of Cells metric is the proportion of cells that have genotype information available. For example, a threshold of 80% retains only variants for which information is available in at least 80% of all cells. Variants with information in fewer than 80% of cells are removed

We recommend using a value of > 50%.

(6) Remove variants mutated in < X of cells

The Variants Mutated in Min % of Cells metric is the percentage of cells across all cells with a genotype call that have a non-references genotype call (e.g., heterozygous or homozygous alternate).

The select threshold is dependent on the sample type and the scientific question one intends to address. For instance rare subclone detection require a lower threshold.

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Review Variants

Navigation Bar – Filter Variants

Menu Description
Search variants Search variants with keywords (e.g., TP53)
Function Annotations List variants that are in coding, intronic, splicing regions. Selecting ‘Blank’ lists variants with no available database annotation information.
Impact Annotations List variants that are frameshift, missense, synonymous. Selecting ‘Blank’ lists variants with no available database annotation information.
ClinVar Annotations List variants with ClinVar annotations. Selecting ‘Blank’ lists variants with no available database annotation information.
White List List variants that are listed in an optionally uploaded white list (BED) file. Selecting False lists all variants. Deselecting False only lists variants found in the white list.
All Variants Resets above filters and lists all variants.
Selected Variants List variants that are checkmarked in the Variant Table.

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Navigation Bar – Filter Subclones

Menu Description
Ignore zygosity
DEFAULT = unchecked
Disregards mutant genotype information. Cells with heterozygous and homozygous genotype calls are treated equally.
Remove subclones with < 1.00 % of cells
DEFAULT = checked
Removes subclones with select percentage of cells from Subclone table. All removed subclones are listed as Small Subclones in the Subclone table.
Remove subclones with missing genotypes
DEFAULT = checked
Removes subclones that contain missing genotypes (NA) in at least one selected variant used for subclone identification.
Show VAF
DEFAULT = checked
Lists the average variant allele frequencies per variant per subclone across all loaded samples in the Subclone Table.

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Navigation Bar – Selected Data


 
  • Lists the total number of variants that are selected in the main window.
  • Lists the total number of subclones defined by all selected variants.

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Main Window – Variants

Variant <Gene Name>_<Amplicon>:<Chr>:<Pos>:<Allele>
Function Function Annotations (e.g., coding)
Protein Protein nomenclature of variant
Coding impact Impact Annotations (e.g., missense)
ClinVar Annotations from ClinVar version 20170801
‘.’ indicates no associated ClinVar entry
‘1’ indicates one associated ClinVar entry
DANN DANN score with a range of 0 (low pathogenicity) – 1 (high pathogenicity).
Whitelist Logical value that indicates whether a variant is included in an optionally uploaded white list (TRUE) or not (FALSE).
 # of Genotyped Cells
  • Number and percentage of filtered cells across all samples with genotype information.
  • Number and percentage of filtered cells across all samples with mutant genotype calls (heterozygous, homozygous).
Sample Column
[Sample 1]
  • Number and percentage of filtered cells across [Sample 1] with genotype information.
  • Number and percentage of filtered cells across [Sample 1] with mutant genotype calls (heterozygous, homozygous)
  • Aggregated variant allele frequency (VAF) of filtered cells across [Sample 1] based on genotype information (diploidy assumed)
  • Aggregated VAF of filtered cells across [Sample 1] based on sequencing read information (comparable to bulk sequencing)
  • The Subclones table may be exported by clicking the icon on the top right corner of the table:  
Additional information about each variant can be found by highlighting the variant in the table (FLT3:p.D835V in image) and review the variant summary to the right side of the table. Clicking the blue hyperlink will provide additional annotation information via the VARSOME database.

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Main Window – Annotations

 

The Annotations window provides a list of databases that Tapestri Insights accesses. General information about each database can be accessed by clicking on the blue hyperlink in the Name column.

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Main Window – Subclones

The Subclone table is segmented into two sections:

  • The top section summarizes the number of subclones that are identified across all loaded samples. The cluster identity and cluster number changes every time the variant list is updated. Percentages shown in parenthesis after each cluster-variant pair denote the average VAF % across all loaded samples.
  • The bottom section summarizes the relative fractions of subclones per loaded sample. Integer numbers represent the number of cells per variant per subclone. Please note that subclone percentage numbers are relative and include subclones that are grouped as “Small Subclones” and “Missing GT Subclones”.

To assess relative subclone size within and across different samples we recommend reviewing the data in the REVIEW SUBCLONES page.

Variant Lists all selected variants as <Gene Name>_<Amplicon>:<Chr>:<Pos>:<Allele>
C1 Subclone 1. Subclone-associated variants are either WT, Het, Hom (zygosity included) or WT, Mut (zygosity ignored). NA represent missing genotype (GT) information.
The percentage in parentheses (Show VAF) reflect the average VAF of a given variant in that clone across all loaded samples.
Small Subclones (10) 10.00 % All subclones with small percentages summarized. Number in parentheses represents total number of Small Subclones. Percentage represents total fraction of Small Subclone-cells out of all cells pass-filter.

Missing GT Subclones (10) 10.00%
All subclones with a missing value (NA) in at least one selected variant summarized. Number in parentheses represents total number of Subclones with missing GT information. Percentage represents total fraction of Missing GT Subclones-cells out of all cells pass-filter.
  • Each subclone is labeled as C1, C2, etc. Subclones may be renamed by double-clicking in the column header of the subclone.
  • The Subclones table may be exported by clicking the icon on the top right corner of the table:

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Main Window – Variant Viewer

The Variant Viewer provides additional quantitative metrics for each variant to help assess its quality and validity. Information of a variant that is highlighted in the Variants table will be shown in this section.

In particular variant-specific allele frequency distributions may be reviewed either across a set of samples, subclones, or both. Each dot represent a cell. Data may be color-coded by sample or subclone. (1) Allele Frequency (VAF), (2) Genotype Quality or (3) Read Depth may be plotted as a function of either Subclone or Sample.

Y-axis Select metric to be plotted against the y-axis of the Variant Viewer plot: Allele Frequency, Quality, Read Depth
Color Select metric to be used to color the data: Sample, Subclone
Violin distribution Checkbox to either display or not display violin plots on top of data.

Mean (red)
Checkbox to either display or not display the mean of single-cell VAFs per plot.

Median (green)
Checkbox to either display or not display the median of single-cell VAFs per plot.

Sub-sample cell
Checkbox to either subsample the data (n = 500 for each group) or use all data for visualization purposes.
  • The Variant Viewer plot may be exported by clicking the icon on the top right corner of the window:  . Supported file formats include PNG, SVG, and PDF.

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Review Subclone Structure

View Options

Group by Sample Visualizes subclones on a per-sample basis. Individual subclones are color-coded differently.
Group by Subclone Visualizes subclones on a per-subclone basis. Individual samples are color-coded differently.
Show percentage Visualizes the subclones on a fractional basis from 0 – 100%. If unchecked subclones are shown on a cell-number basis.
Show Labels Adds labels to each subclone.

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Show Samples

  • Lists all samples that are loaded.
  • Samples may be selected or deselected by highlighting them with the mouse cursor.

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Show Subclones

  • Lists all subclones that are identified with all selected variants.
  • Subclones may be selected or deselected by highlighting them with the mouse cursor.

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Subclones (Graphic)

  • Visual representation of subclones in boxplot format.
    Each boxplot refers to either (1) one of the loaded sample visualizing different-colored subclones (Group by Sample option) or (2) one subclone visualizing different-colored samples (Group by Subclone option).
  • The Subclones image may be exported as a high-resolution image by clicking the icon on the top right corner of the image . Supported file formats include PNG, SVG, and PDF.

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Subclones (Table)

The Subclonetable is identical to the table that is displayed in the REVIEW VARIANTS page.

  • The top section summarizes the number of subclones that are identified across all loaded samples. The cluster identity and cluster number changes every time the variant list is updated. Percentages shown in parenthesis after each cluster-variant pair denote the average VAF % across all loaded samples.
  • The bottom section summarizes the relative fractions of subclones per loaded sample. Integer numbers represent the number of cells per variant per subclone. Please note that subclone percentage numbers are relative and include subclones that are selected inside the Show Subclones field.
  • The Subclone table may be exported by clicking the icon on the top right corner of the image

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Release Notes

Release Notes

Tapestri Insights v1.6.1

  • Bug fixes

Tapestri Insights v1.6.0

  • Addition of new visualizes:
    • Insightful visualizations of variants across samples and subclones
    • (Beta) Heatmap for visualization of many variants across subclones
    • (Beta) Fish plot for visualization of subclones over time
  • Analysis results will be identical to Tapestri Insights v1.4.2
  • Annotation cache
  • Loading & Filtering speedup
  • Update checking can be turned off in preferences

Tapestri Insights v1.4.3

  • Maintenance release with minor bug fixes and user interface improvements
  • Analysis results will be identical to Tapestri Insights v1.4.2

Tapestri Insights v1.4.2

  • Multi-sample support – Track subclones across samples, even when variants/subclones are not present in some samples
    • VCF and LOOM files now contain records for all sites
      • VCF and LOOM files significantly larger due to the addition of all sites
    • Existing LOOM files are still compatible, but for multi-sample analysis rerunning analysis is required
  • Improved variant annotations – New annotation service enable access to over 30 public genomic databases to quickly identify relevant variants
  • Ability to export filtered cells and variants data to CSV or loom files enables additional analysis outside of Tapestri Insights

 

Tapestri Insights v1.0.2

  • The first release!
  • Load your single cell DNA data
  • Search and select relevant variants
  • Explore subclones
    • Co-occurrence of mutations
    • Rare populations
  • Save and share analysis
    • Save analysis as .TAP file for sharing and record keeping
    • Export tables and figures

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