Motif-based Feature Extraction
Command: krewlyzer motif
Purpose
Extracts end motif, breakpoint motif, and Motif Diversity Score (MDS) from sequencing fragments.
Biological Context
Motif analysis of cfDNA fragment ends can reveal tissue-of-origin, nucleosome positioning, and nuclease activity. MDS quantifies motif diversity, which may be altered in cancer. See Zhou et al., 2020 for details on jagged ends and nuclease biology.
Usage
krewlyzer motif /path/to/input.bam -g /path/to/reference.fa -o /path/to/output_dir \
-k 4 --threads 4
Output
{Sample}.EndMotif.tsv: Frequency of K-mer sequences at fragment 5' ends.{Sample}.BreakPointMotif.tsv: Frequency of K-mer sequences flanking the breakpoint (context + fragment start).{Sample}.MDS.tsv: Motif Diversity Score (Shannon entropy of end motifs).
Method
Motif frequencies are calculated by analyzing the K-mer sequences at the 5'-ends of fragments. - End Motif: Raw sequence at the end of the fragment. - Breakpoint Motif: Sequence context from the reference genome flanking the fragment start point. - MDS: Normalized Shannon entropy of the End Motif distribution.
Calculation Details
Motif Diversity Score (MDS)
MDS quantifies the randomness of the 4-mer end motifs.
$$ MDS = \frac{ - \sum_{i} p_i \log_2(p_i) }{ \log_2(4^k) } $$
Where: - $p_i$ is the frequency of the $i$-th motif. - $k$ is the k-mer length (default 4). - The denominator normalizes the score to $[0, 1]$.
Clinical Interpretation
MDS Values
| MDS | Interpretation |
|---|---|
| High (~1.0) | Random/diverse fragmentation (healthy-like) |
| Low | Stereotyped fragmentation (possible tumor signal) |
Healthy vs Cancer
| Metric | Healthy | Cancer (ctDNA) |
|---|---|---|
| MDS | Higher (random) | Lower (stereotyped) |
| Jagged ends | Lower | Higher |
| Specific motifs | Baseline | Enriched cancer-associated motifs |
Biological Basis
- cfDNA fragmentation is driven by nucleases (DNASE1, DNASE1L3)
- ~87.8% of cfDNA molecules have jagged (single-stranded) ends
- Tumor-derived fragments show higher jaggedness than wild-type
Reference: See Citation & Scientific Background for detailed paper summary.