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87 lines
4.1 KiB
Markdown
87 lines
4.1 KiB
Markdown
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# Obiclean: PCR Amplicon Error Correction & Chimera Detection
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Obiclelan is a Go package for cleaning high-throughput amplicon sequencing data. It corrects PCR/sequencing errors by leveraging abundance-weighted sequence relationships and optionally detects chimeric artifacts using graph-based heuristics. Built for scalability, it integrates with OBITools4’s data model and supports IUPAC ambiguity codes.
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## Core Concepts
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- **`seqPCR`**: Represents a sequence in one sample, with fields for raw count (`Count`) and post-clustering weight (`Weight`), plus graph edges, annotations, and cluster membership.
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- **Directed similarity graphs**: Edges point from more abundant (father) to less abundant (son) sequences differing by ≤ *d* nucleotides.
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- **Abundance-weighted correction**: Less abundant sequences are penalized unless supported by strong graph evidence.
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---
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## Public Functionalities
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### 1. **Graph Construction**
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- `BuildSeqGraph(samples, distance)`: Builds a mutation graph across samples.
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- Compares all sequence pairs within/between samples.
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- Adds directed edges only if father has higher weight and differs by ≤ `distance` mismatches.
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- Uses parallel workers (`buildSamplePairs`) for one-error edges and `FastLCSScore` for multi-error extensions.
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- `FilterGraphOnRatio(samples, ratio)`: Removes spurious edges violating a power-law decay model:
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`weight_ratio < (ratio)^distance`. Ensures only statistically plausible edges remain.
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---
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### 2. **Annotation & Status Assignment**
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- `annotateOBIClean(samples)`: Populates per-sequence annotations:
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- `"obiclean_head"`: `true` if the sequence has no incoming edges (i.e., is a cluster head).
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- `"obiclean_singletoncount"`, `"internalcount"`, `"headcount"`: Global counts of sequences in each status across all samples.
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- `ObicleanStatus(seq) string`: Returns one of:
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- `"s"`: Singleton (no edges).
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- `"h"`: Hub (has outgoing → sons, but no incoming father) — likely erroneous ancestor.
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- `"i"`: Internal (has both parents and children) — intermediate error variant.
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- `Status(seq, sample)` / `Weight(seq, sample)`: Get/set per-sample status (`h/i/s`) and weight annotations.
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---
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### 3. **Clustering & Head Selection**
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- `GetCluster(seq, sample)`: Retrieves or initializes cluster membership (e.g., `"cluster_42"`).
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- `GetMutation(seq) map[string]int`: Returns mutation counts (e.g., `"A->T@42": 3`).
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- `Mutation(samples)`: Populates mutation annotations from graph edges.
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---
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### 4. **Chimera Detection**
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- `AnnotateChimera(samples)`: Flags chimeric sequences per sample:
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- Filters candidates to *head* sequences only.
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- For each candidate `s`, scans more abundant parents for prefix/suffix matches:
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- Uses IUPAC-aware comparisons (`commonPrefix`, `commonSuffix`).
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- Skips near-identical pairs (one edit difference via `oneDifference`).
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- Flags as chimera if:
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```
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maxPrefixLen + maxSuffixLen ≥ L
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AND not fully contained in one parent (maxSuffix < L)
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```
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- Annotation format:
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`"parent_left/parent_right@(overlap)(start)(end)(len)"`.
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---
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### 5. **Filtering & Output Control**
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- CLI-style filters (applied post-processing):
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- `OnlyHead`: Keep only `"obiclean_head"` sequences.
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- `NotAlwaysChimera`: Exclude sequences flagged chimera in *all* samples.
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- `MinSampleCount(n)`: Retain sequences present ≥ *n* times across samples.
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- Optional exports:
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- `SaveGMLGraphs(samples)`: Writes per-sample graphs in GML (node shapes/colors encode abundance/status).
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- `EmpiricalDistCsv(samples)`: Exports substitution statistics (e.g., A→C rates at position *i*) to compressed CSV.
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- `EstimateRatio(samples, minStatCount)`: Collects distance-1 substitution events for downstream modeling.
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---
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## Design Highlights
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- **IUPAC-compliant comparisons**: Nucleotide equality via `obiseq.SameIUPACNuc`.
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- **Annotation-driven**: No in-place mutation; all metadata stored via `BioSequence.Annotations`.
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- **Scalable parallelism**: Uses goroutines + channels for pairwise comparisons; integrates `progressbar`/Logrus.
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- **Flexible thresholds**: Configurable via flags (`distance`, `ratio`, `min-sample-count`), defaulting to sensitivity-optimized values.
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