Preseq

A shallow curve indicates complexity saturation. The dashed line shows a perfectly complex library where total reads = unique reads.

When preseq lc_extrap is run with the default parameters, the extrapolation points reach 10 billion molecules making the plot difficult to interpret in most scenarios. It also includes a lot of data in the reports, which can unnecessarily inflate report file sizes. To avoid this, MultiQC trims back the x-axis until each dataset shows 80% of its maximum y-value (unique molecules).

To disable this feature and show all the data, add the following to your MultiQC configuration:

preseq:
  notrim: true

Using coverage instead of read counts

Preseq reports its numbers as “Molecule counts”. This isn’t always very intuitive, and it’s often easier to talk about sequencing depth in terms of coverage. You can plot the estimated coverage instead by specifying the reference genome or target size, and the read length in your MultiQC configuration:

preseq:
  genome_size: 3049315783
  read_length: 300

These parameters make the script take every molecule count and divide it by (genome_size / read_length).

MultiQC comes with effective genome size presets for Human and Mouse, so you can provide the genome build name instead, like this: genome_size: hg38_genome. The following values are supported: hg19_genome, hg38_genome, mm10_genome.

When the genome and read sizes are provided, MultiQC will plot the molecule counts on the X axis (“total” data) and coverages on the Y axis (“unique” data). However, you can customize what to plot on each axis (counts or coverage), e.g.:

preseq:
  x_axis: counts
  y_axis: coverage

Plotting externally calculated read counts

To mark on the plot the read counts calculated externally from BAM or fastq files, create a file with preseq_real_counts in the filename and place it with your analysis files. It should be space or tab delimited with 2 or 3 columns (column 1 = preseq file name, column 2 = real read count, optional column 3 = real unique read count). For example:

Sample_1.preseq.txt 3638261 3638011
Sample_2.preseq.txt 1592394 1592133
[...]

You can generate a line for such a file using samtools:

echo "Sample_1.preseq.txt "$(samtools view -c -F 4 Sample_1.bam)" "$(samtools view -c -F 1028 Sample_1.bam)

File search patterns

preseq:
  - contents: EXPECTED_DISTINCT
    num_lines: 2
  - contents: distinct_reads
    num_lines: 2
preseq/real_counts:
  fn: "*preseq_real_counts*"