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Introduction

Soft-clipping of reads is done during alignment to mask portions of the reads that do not align to the reference sequence.

Visualizing soft-clipping can be useful for identifying contaminating adapter sequences or detecting structural variants. 

How to view soft-clipping in IGB

Note: Soft-clipped sections of reads are not visible in versions of IGB prior to 9.1

To view your own data with soft-clipping in IGB:

  1. Add your BAM file to IGB.
  2. Zoom in on the gene or region of interest.
  3. Click Load Data.

To view the example soft-clipping data in the images below:

  1. Open the human genome (H_sapiens_Dec_2013) in IGB.
  2. Navigate to: chr1:3,320-26,768
  3. Hide the RefSeq Curated annotation track by right-clicking and selecting Hide.
  4. Download the two files below:
    1. pacBio.bam
    2. pacBio.bam.bai
  5. In IGB, select File > Open File...
  6. Select the pacBio.bam file and click Open.
  7. Click Load Sequence to load sequence data.
  8. Click Load Data to load data into it.

 

Soft-clipping is enabled by default in IGB, with the soft-clipped section of the read displayed in gray.

To configure the appearance of soft-clips:

  1. Right-click the reads track label.
  2. Select Configure soft-clip.

Options for viewing soft-clipping include:

  1. Show as default color - Soft-clipping shows as a default gray.
  2. Show as custom color... - Using a color picker, soft-clipping shows as selected color.
  3. Show as bases - Soft-clipping shows as Residue Colors.
  4. Hide soft-clipping - Soft-clipping is hidden.

 

 

 

Note: When using the hide soft-clipping option, a line showing the full length of the read including the soft-clipped portion will appear.

To completely hide the soft-clipping portion of the read, use the Show as Custom Color soft-clip option, and select the same color as the background for the track.

Track Operations and Soft-clipping

Track Operations behave differently when soft-clipping is present.

  • Depth Graph (All) and Depth Graph (Start) ignore the soft-clipped parts of reads.
  • Mismatch Graph and Mismatch Pileup Graph include the soft-clipped parts of reads.

 

Data used in these examples is a subset taken from the Genome in a Bottle consortium (HG002 PacBio CCS 10kb):

Zook JM, Catoe D, McDaniel J, et al. Extensive sequencing of seven human genomes to characterize benchmark reference materials. Sci Data. 2016;3:160025. Published 2016 Jun 7. doi:10.1038/sdata.2016.25

 

 

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