Illumina 5-Base

Illumina 5-Base (the “5-Base DNA Prep”) is the chemical inverse of bisulfite. The enzyme converts 5-methylcytosine to thymine and leaves unmethylated cytosine intact, so the library keeps its full complexity and the raw reads align to the unconverted genome with a standard aligner. Bismark then calls methylation at inverted polarity: a read T at a genomic C is methylated (Z), a read C is unmethylated (z).

The mode is opt-in behind --illumina_5base and is paired-end only (the 5-Base library is paired-end and directional; single-end input is rejected at startup).

Stability contract

5-Base is not byte-identical to anything: Perl Bismark has no 5-Base path, so there is no byte-for-byte oracle. It is instead concordance-gated, the same contract as the --rammap and --combined_index paths:

• Supported in GA (3.0.0): --illumina_5base (core), --five_base_duplex, --five_base_consensus, --five_base_consensus_from_bam, --five_base_deconvolution. Each is validated against Illumina DRAGEN on the real NA12878 demo at full depth by the metric appropriate to what it produces: core by per-CpG concordance with DRAGEN’s (deduplicated, full-depth) CX report (deduplicated, iso-DRAGEN) — r approximately 0.99 over 55M CpGs (0.998 at cov>=10), 99.3% call-agreement at cov>=10 (98.8% at cov>=1); deconvolution by variant precision/recall vs DRAGEN’s germline VCF — 90.3% / 93.4%; duplex / consensus (per-molecule collapse — DRAGEN builds a duplex-consensus methylation track only for UMI/enrichment kits, so there is no WGS consensus-CX to correlate against) by per-strand mean-methylation agreement with DRAGEN (~47.9% vs ~48%, bias-free). All four are additionally held to a reproducible lambda/pUC19 spike-in gate that runs in CI with no proprietary data.
• Preview: --five_base_umi_qname (duplex UMI taken from the read name). It works and is exercised on real data, but is not yet held to the same gated contract.
• Unchanged: the faithful bisulfite / EM-seq suite is byte-frozen. Every legacy methylation call still reduces to the exact Perl v0.25.1 output (the perl-oracle CI gate stays green); turning 5-Base on never perturbs a bisulfite run.

Because it is concordance-gated rather than byte-identical, every 5-Base path is opt-in and never silent: an unsupported combination (single-end, --non_directional, --pbat, --combined_index) is rejected loudly rather than silently degraded.

Running it

Directional, paired-end, against the unconverted genome. minimap2 (-x sr) against the genome FASTA is the default engine; bowtie2 or hisat2 work against a NORMAL (unconverted) index supplied with --five_base_index.

# core: align + inverted-polarity methylation calls
bismark --illumina_5base --genome /path/to/GRCh38 -1 R1.fastq.gz -2 R2.fastq.gz

# bowtie2/hisat2 instead of minimap2 (build a plain index once with bowtie2-build)
bismark --illumina_5base --bowtie2 --five_base_index /path/to/normal_idx \
        --genome /path/to/GRCh38 -1 R1.fastq.gz -2 R2.fastq.gz

The emitted BAM is standard Bismark convention (XM/XR/XG), so the extractor, bismark2bedGraph, coverage2cytosine, and the reports consume it unchanged.

Advanced modes

Real 5-Base libraries carry a dual UMI in the read name and sequence each molecule as two strand-partner read pairs. These modes use that structure:

• --five_base_deconvolution separates a genuine C>T/G>A genetic variant from 5mC using both strands (DRAGEN’s rule: methylation moves only one strand, a variant moves both). Writes a per-CpG report; variant CpGs are excluded from the methylation totals.
• --five_base_duplex groups the two strands of each molecule into a duplex family (keyed on the fragment span plus the canonical dual UMI) and reconciles the 5mC signal per molecule.
• --five_base_consensus collapses each duplex family to one consensus record per molecule, scoring both strands of every CpG.
• --five_base_consensus_from_bam re-runs the duplex-consensus collapse over one or more already-aligned 5-Base BAMs (same output as --five_base_consensus, skipping re-alignment).
• --five_base_umi_qname (preview) takes the duplex UMI from the read name (the real Illumina form, A+B with the partner swapped) instead of an inline read prefix.

# duplex consensus from the real dual-UMI in the read name
bismark --illumina_5base --five_base_umi_qname --five_base_consensus \
        --genome /path/to/GRCh38 -1 R1.fastq.gz -2 R2.fastq.gz

Validation

The concordance evidence (per-CpG Pearson r and call-agreement vs DRAGEN, and the deconvolution precision/recall vs DRAGEN’s germline VCF) and a reproducible runbook live in the repository at validation/VALIDATION_REAL_DATA.md, with the validation/concordance.py harness. The deterministic floor (lambda unmethylated control reads near 0%, pUC19 CpG-methylated control reads high) is locked by the five_base_controls_* gates that run in CI.