---
title: "The case of the missing mussel: recovering a lost sample and mapping the mussel methylome"
author: "Steven Roberts"
date: "2026-07-08"
categories: [notebook, methylation, Mytilus, WGBS]
format:
  html:
    toc: true
    toc-depth: 2
    code-fold: true
    fig-cap-location: bottom
---

## Why we're looking at mussels

Bay mussels (*Mytilus trossulus*) spend their whole lives filtering seawater in
one spot, so they end up as living records of whatever is in the water around
them. That makes them excellent **sentinels** for coastal pollution. In this
project we're studying mussels from Puget Sound that grew at six different
locations — three relatively clean sites and three with high levels of
**PAHs** (polycyclic aromatic hydrocarbons, a family of pollutants that come
from oil, soot, and combustion). Twenty-four mussels in all, four from each
site.

The question behind the whole project is: **does pollution leave a readable mark
on a mussel's DNA?** Not by changing the DNA letters themselves, but through
*DNA methylation* — small chemical tags that sit on top of the DNA and can turn
the volume of genes up or down. If polluted-site mussels carry a consistent,
distinctive methylation pattern, that pattern could become a **biomarker**: a
cheap, fast test for exposure that doesn't require expensive chemistry on the
water itself.

This post is about a piece of housekeeping that turned into a small detective
story, plus the first big-picture look at what mussel DNA methylation actually
looks like.

## A quick word on the data

To read methylation across the whole genome we use **whole-genome bisulfite
sequencing (WGBS)**. The practical upshot: for each mussel we get a giant table
listing millions of individual **CpG sites** (specific spots in the DNA where
methylation happens) and, for each one, what fraction of DNA copies carried the
methyl tag. One mussel's table is about **750 megabytes**. There are 24 of them.

## The mystery: one mussel had no data

While setting up the analysis, one sample — mussel **93M**, from the Smith Cove
industrial site — was missing its data file. At first glance the project notes
suggested 93M had been dropped earlier as a "low-quality outlier," so a missing
file seemed plausible.

But something didn't add up. The **raw sequencing reads** for 93M were sitting on
our server, and they were a completely normal size — just as big as every other
mussel's. A genuinely low-quality sample would have *fewer* reads, not a full
set. So why was there no finished data?

Digging into the records revealed the answer: 93M had **never actually been
processed**. What existed was a tiny **10,000-read test run** — the kind of
quick check you do to make sure a pipeline works before turning it loose on the
real thing. That test had quietly been mistaken for the finished product. The
"outlier" was never an outlier; it was simply **unfinished**.

## The recovery

Fixing it meant running 93M through the exact same assembly line as the other 23
mussels, from raw reads to finished methylation table:

1. **Line up ~41 million pairs of DNA reads** against the mussel reference
   genome (the slow step — about a day of computing).
2. **Remove duplicate reads** left over from sample prep.
3. **Read off the methylation** at every covered CpG site.
4. **Merge the two DNA strands** into one tidy per-site table.

The result: a complete, standard-sized file for 93M with **17.2 million CpG
sites** and an overall methylation level of **11.65%** — right in line with the
other mussels. A sample that had been written off was now a full, usable member
of the group. (There were a few potholes along the way — a blocked download
here, an incompatible software setting there — but nothing that a second pass
couldn't fix.)

## What does a mussel methylome look like?

With all 24 mussels finally in hand, we could draw the first complete picture of
the *M. trossulus* methylation "landscape." Four features stand out, and
reassuringly, they all match what's known from other shellfish.

### 1. Most of the genome is *un*-methylated

Averaged across the mussels, only about **11–12%** of CpG sites carry a methyl
tag. Human DNA, by comparison, is methylated at most of its CpG sites. Low,
sparse methylation is the normal state for invertebrates like mussels and
oysters.

### 2. It's an on-or-off, "mosaic" pattern

Methylation in mussels isn't a smooth dial — it's more like a light switch.
Sites tend to be either almost fully off or almost fully on, with relatively few
in between:

| Methylation state | Share of sites |
|---|---:|
| Off (unmethylated) | **80%** |
| In-between | 10% |
| On (methylated) | 10% |

![Genome-wide methylation is bimodal: most sites cluster near "off," with a
smaller peak near "on."](../output/27-methylation-landscape/bimodality_pooled.png){width=70%}

### 3. Methylation lives inside genes, not around them

Where the methyl tags *do* land is highly non-random. They're concentrated in
the **bodies of genes** and largely absent from the switch-like regions
(promoters) and the empty stretches between genes:

| Part of the genome | Average methylation |
|---|---:|
| Gene bodies (genes, exons, introns) | **~20%** |
| Promoters (gene "on-switches") | 7% |
| Between genes (intergenic) | 5% |

![Methylation is enriched in gene bodies and depleted in promoters and
intergenic space — the classic molluscan "gene-body methylation"
pattern.](../output/28-genomic-feature-distribution/feature_methylation_barplot.png){width=75%}

This "gene-body methylation" is the signature pattern of invertebrates, and it's
thought to mark the steady, housekeeping genes a cell relies on all the time.

### 4. The genome itself remembers where methylation has been

There's an elegant twist here. Methylation is chemically corrosive to DNA over
evolutionary time — spots that have been methylated for millions of years slowly
*lose* their CpG sites. So we can measure how "CpG-depleted" each gene is and use
that as a fossil record of its long-term methylation history.

Sure enough, mussel genes fall into **two camps** — one CpG-rich, one
CpG-depleted — and the CpG-depleted genes are exactly the ones we observe to be
**heavily methylated today** (a strong negative relationship, correlation
≈ −0.68). The genome's ancient history and its present-day chemistry tell the
same story.

![Genes that are CpG-depleted (left) are the ones most heavily methylated today —
linking long-term evolutionary history to the current
methylome.](../output/29-cpg-oe-mosaic/oe_vs_methylation.png){width=70%}

## And about that "outlier"…

Now that 93M was fully processed, we could finally check whether it deserved its
bad reputation. It didn't. Ranked against all 24 mussels by overall methylation,
93M came in **14th — dead center of the pack**. Its recovered data is normal in
every way that matters. Its earlier exclusion was a processing gap, not a
quality problem.

## The bottom line

- A mussel thought to be a low-quality dropout was actually just **never
  finished processing**; it has now been fully recovered and looks completely
  normal.
- Across all 24 Puget Sound mussels, DNA methylation is **low and sparse (~12%)**,
  organized as an **on/off mosaic**, and **concentrated inside genes** — the
  textbook pattern for shellfish.
- The genome's own CpG-depletion "fossil record" lines up with the methylation
  we measure today.

This is the descriptive baseline. The next chapter asks the question that
started it all: **do the mussels from polluted sites carry methylation
differences from the clean-site mussels** — and could any of those differences
serve as a practical pollution biomarker? That comparison is where we head next.

---

*Technical note: this landscape was produced with methylKit and BEDTools from
merged-CpG coverage files; scripts and full results are in `code/26`–`code/29`
and `output/26`–`output/29` of the project repository. The 93M recovery
pipeline is `code/25.1-93M-cov-recovery.sh`.*
