Visualizing and Investigating Short-Read Alignments

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When to Use the NGS Browser to Visualize and Investigate Data Opening the NGS Browser Importing Data into the NGS Browser Zooming and Panning to a Specific Region of the Alignment Viewing Coverage of the Reference Sequence Viewing the Pileup View of Short Reads Comparing Alignments of Multiple Data Sets Viewing Location, Quality Scores, and Mapping Information Flagging Reads Evaluating and Flagging Mismatches Viewing Insertions and Deletions Viewing Feature Annotations Printing and Exporting the Browser Image

When to Use the NGS Browser to Visualize and Investigate Data

The NGS Browser lets you visually verify and investigate the alignment of short-read sequences to a reference sequence, in support of analyses that measure genetic variations and gene expression. The NGS Browser lets you:

• Visualize short-read data aligned to a nucleotide reference sequence.

• Compare multiple data sets aligned against a common reference sequence.

• View coverage of different bases and regions of the reference sequence.

• Investigate quality and other details of aligned reads.

• Identify mismatches due to base calling errors or polymorphisms.

• Visualize insertions and deletions.

• Retrieve feature annotations relative to a specific region of the reference sequence.

• Investigate regions of interest in the alignment, determined by various analyses.

You can visualize and investigate the aligned data before, during, or after any preprocessing (filtering, quality recalibration) or analysis steps you perform on the aligned data.

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Opening the NGS Browser

To open the NGS Browser, type the following in the MATLAB Command Window:

ngsbrowser

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Importing Data into the NGS Browser

Browser Displaying Reference Track, One Alignment Track, and One Annotation Track

Importing a Reference Sequence

You can import a single reference sequence into the NGS Browser. The reference sequence must be in a FASTA file.

1. Select File > Add Data from File.

2. In the Open dialog box, select a FASTA file, and then click Open.

Tip   You can use the getgenbank function with the ToFile and SequenceOnly name-value pair arguments to retrieve a reference sequence from the GenBank database and save it to a FASTA-formatted file.

Importing Short-Read Alignment Data

You can import multiple data sets of short-read alignment data. The alignment data must be in either of the following:

• BioMap object

  Tip   Construct a BioMap object from a SAM- or BAM-formatted file to investigate, subset, and filter the data before importing it into the NGS Browser.

• SAM- or BAM-formatted file

  Note   Your SAM- or BAM-formatted file must: Have reads ordered by start position in the reference sequence. Have an IDX index file (for a SAM-formatted file) or BAI and LINEARINDEX index files (for a BAM-formatted file) stored in the same location as your source file. Otherwise, the source file must be stored in a location to which you have write access, because MATLAB needs to create and store index files in this location.

  Tip   MathWorks recommends using SAMtools to check if the reads in your SAM- or BAM-formatted file are ordered by position in the reference sequence, and also to reorder them, if needed.

  Tip   If you do not have index files (IDX or BAI and LINEARINDEX) stored in the same location as your source file, and your source file is stored in a location to which you do not have write access, you cannot import data from the source file directly into the browser. Instead, construct a BioMap object from the source file using the IndexDir name-value pair argument, and then import the BioMap object into the browser.

To import short-read alignment data:

1. Select File > Add Data from File or File > Import Alignment Data from MATLAB Workspace.

2. Select a SAM-formatted file, BAM-formatted file, or BioMap object.

3. If you select a file containing multiple reference sequences, in the Select Reference dialog box, select a reference or scan the file for available references and their mapped reads counts. Click OK.

4. Repeat the previous steps to import additional data sets.

Importing Feature Annotations

You can import multiple sets of feature annotations from GFF- or GTF-formatted files that contain data for a single reference sequence.

1. Select File > Add Data from File.

2. In the Open dialog box, select a GFF- or GTF-formatted file, and then click Open.

3. Repeat the previous steps to import additional annotations.

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Zooming and Panning to a Specific Region of the Alignment

To zoom in and out:

Use the toolbar buttons,

or click-drag an edge of the rubberband in the Overview area.

To pan across the alignment:

Use the toolbar buttons,

or click-drag the rubberband in the Overview area.

Tip   Use the left and right arrow keys to pan in one base pair (bp) increments.

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Viewing Coverage of the Reference Sequence

At the top of each alignment track, the coverage view displays the coverage of each base in the reference sequence. The vertical ruler on the left edge of the coverage view indicates the maximum coverage in the display range. Hover the mouse pointer over a position in the coverage view to display the location and counts.

Note   The browser computes coverage at the base pair resolution, instead of binning, even when zoomed out.

To change the percent coverage displayed, click anywhere in the alignment track, and then edit the Alignment Coverage settings.

Tip   Set Max to a value greater than 100, if needed, when comparing the coverage of multiple tracks of reads.

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Viewing the Pileup View of Short Reads

Each alignment track includes a pileup view of the short reads aligned to the reference sequence.

Limit the depth of the reads displayed in the pileup view by setting the Maximum display read depth in the Alignment Pileup settings.

Tip   Limiting the depth of short reads in the pileup view does not change the counts displayed in the coverage view.

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Comparing Alignments of Multiple Data Sets

Compare multiple data sets, with each data set in its own track, against a common reference sequence. Use the Track List to show/hide, order, and delete tracks of data.

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Viewing Location, Quality Scores, and Mapping Information

Hover the mouse pointer over a position in a read to display strand direction, location, quality, and mapping information for the base, the read, and its paired mate.

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Flagging Reads

Click anywhere in an alignment track to display the Alignment Pileup settings.

Flagging Reads with Low Mapping Quality

Set the Mapping quality threshold in the Alignment Pileup section to flag low-quality reads. Reads with a mapping quality below this level appear in a lighter shade of gray.

Flagging Duplicate Reads

Select Flag duplicate reads and select an outline color.

Flagging Reads with Unmapped Pairs

Select Flag reads with unmapped pair and select an outline color.

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Evaluating and Flagging Mismatches

Mismatches display as colored blocks or letters, depending on the zoom level.

Zoomed out view of read — Mismatches display as bars

Zoomed in view of read — Mismatches display as letters

In addition to the base Phred quality information that displays in the tooltip, you can visualize quality differences by using the Shade mismatch bases by Phred quality settings.

The mismatch blocks or letters display in:

• Light shade — Mismatch bases with Phred scores below the minimum

• Graduation of medium shades — Mismatch bases with Phred scores within the minimum to maximum range

• Dark shade — Mismatch bases with Phred scores above the maximum

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Viewing Insertions and Deletions

The NGS Browser designates insertions with a symbol. Hover the mouse pointer over the insertion symbol to display information about it.

The NGS Browser designates deletions with dashes.

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Viewing Feature Annotations

After importing a feature annotation file, you can zoom and pan to view feature annotations associated with a region of interest in the alignment. Hover the mouse pointer over the feature annotation.

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Printing and Exporting the Browser Image

Print or export the browser image by selecting File > Print Image or File > Export Image.

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Storing and Managing Feature Annotations in Objects

Identifying Differentially Expressed Genes from RNA-Seq Data

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