Computational validation report
Full-reference FASTQ-to-BAM fusion replay
Seven matched controls were processed through pinned BWA-MEM alignment against whole hg19, followed by FusionSieve analysis of the resulting BAM files.
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Introduction and summary
An earlier audit found that fusion reads inserted directly into BAM files lacked alignment
scores such as AS and XS and used a different split-alignment structure
from nearby BWA-MEM records. A subsequent targeted FASTQ experiment recovered all seven planted
fusions, but it did not create whole-genome alignments. This study performed the missing step:
paired FASTQs were aligned against the full hg19 reference with the recorded historical BWA-MEM
version and -M behavior, sorted and indexed, and then analyzed as BAMs by FusionSieve.
Exactly one target-window candidate was found in each of seven spiked controls, while none was
found in the seven matched backgrounds. All 1,106 planted primary read records mapped. BWA-MEM
generated AS, XS, NM, MD, and read-group tags on
every mapped planted primary record. The maximum absolute breakpoint error was 2 bp.
The full-reference replay solved the alignment-realism defect identified in the direct BAM experiment: the aligner, rather than the spike-in program, calculated the coupled alignment fields. It also reproduced candidate recovery in all seven spiked controls. The calls remain REVIEW-tier research evidence and are not clinical or automatically reportable results.
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Study question and hypotheses
Primary question
Can FASTQ-level fusion generation followed by whole-reference BWA-MEM alignment produce internally coherent synthetic BAM records while preserving matched-background fusion recovery?
Working hypotheses
- Alignment hypothesis: BWA-MEM will calculate alignment scores, edit-distance fields, clipping, flags, mate fields, and split-hit structure from sequence and reference.
- Recovery hypothesis: each spiked BAM will contain one FusionSieve candidate within the predeclared ±5 bp target window.
- Matched-background hypothesis: the corresponding unmodified BAM will contain no candidate in that target window.
- Separation hypothesis: candidate detection and automatic reportability will remain distinct endpoints.
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Experimental design
Seven post-processed BAM backgrounds were represented by neutral control numbers. Five controls received an EML4–ALK design and two received a CD74–ROS1 design. Each control was analyzed twice: once as an unmodified matched background and once after FASTQ-level spike-in. This produced 14 independently aligned and analyzed BAM datasets.
| Component | Design | Purpose |
|---|---|---|
| Matched controls | 7 backgrounds and 7 spiked counterparts | Separate spike-in recovery from pre-existing background calls |
| Fusion definitions | 5 EML4–ALK; 2 CD74–ROS1 | Test two genomic fusion architectures |
| Synthetic dose | 553 read pairs: 420 split fragments and 133 spanning pairs | Provide both junction and mate-pair evidence |
| Alignment analyses | 14 whole-hg19 BWA-MEM runs | Let the aligner calculate coupled BAM fields |
| Primary endpoint | Correct genes, chromosomes, strands, and both breakpoints within ±5 bp | Measure target-window candidate recovery |
| Negative control | Zero target-window candidates in each matched background | Require a paired, background-specific comparison |
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Methods
- Reconstruct paired FASTQ. Complete primary read pairs were recovered from the surviving post-processed BAMs and returned to sequencer orientation. This is a controlled reconstruction, not recovery of the absent instrument FASTQs.
- Remove coordinate-order clustering. Because the source BAMs were coordinate sorted, paired records were deterministically reordered by a pair-preserving HMAC-SHA256 key. The procedure verified matching mates and an unchanged pair multiset. It cannot recreate the original flow-cell order.
- Generate fusion read pairs. SpikeForge created the two fusion designs at the FASTQ level. It did not write BAM alignment scores, flags, CIGAR strings, or mate placements.
-
Align against full hg19. Pinned BWA-MEM 0.7.12-r1039 was run with the historical
-Mconvention, eight threads, and the recorded single read-group structure. The 93-contig hg19 FASTA was locked by SHA-256 and its primary contig names and lengths matched the source BAM headers. - Sort, index, and validate. Samtools 1.24 sorted and indexed each BAM. The workflow checked coordinate order, read-group consistency, index readability, synthetic-read alignment fields, and file hashes before accepting each output.
- Run matched FusionSieve analyses. FusionSieve analyzed all 14 BAMs under the same paired configurations used in the earlier FASTQ study. Candidate calls were compared with truth only after the runs completed.
- Aggregate without sequence data. The public report uses control-level and aggregate statistics only. Read names, source identifiers, local locations, BAMs, FASTQs, and reference indexes are excluded from public artifacts.
Why FASTQ plus BWA is better than assigning tags
Alignment fields are not independent decorations. AS depends on the chosen alignment,
CIGAR, bases, clipping, mismatches, gaps, and scoring parameters. XS describes a
competing alignment found during the reference search and contributes to placement confidence.
MAPQ, NM, MD, SA, flags, and mate fields must describe the same
alignment event. Sampling each field from real-read distributions could reproduce a histogram
while creating impossible individual records. Starting with FASTQ lets one aligner calculate the
fields jointly from the same sequence and reference.
BWA source tag 0.7.12, source commit 5a340797e705d93ef0b0a0f0002eb198a9894e38;
BWA binary SHA-256 a3fb8e9901543ac573e677316aa45c9fc7120c3ff0b88a7ed0c9e34822125309;
hg19 FASTA SHA-256 92b96d16b307d824f3b6b9dc63feb237a75226f82ca2166d51ecec039bee4449.
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Results
5.1 Primary recovery endpoint
All seven target fusions were detected in the spiked BAMs, and all seven matched backgrounds were negative within the locked target windows. Each spiked BAM contained exactly one matching candidate; no target window was ambiguous. The maximum absolute breakpoint error was 2 bp.
| Control | Fusion | Pairs added | Background target | Spiked target | Error (bp) | Unique split/span | Tier |
|---|---|---|---|---|---|---|---|
| Control 1 | EML4–ALK | 80 | 0 | 1 | −2 / +2 | 51 / 20 | REVIEW |
| Control 2 | EML4–ALK | 80 | 0 | 1 | −2 / +2 | 55 / 18 | REVIEW |
| Control 3 | EML4–ALK | 80 | 0 | 1 | −2 / +2 | 56 / 20 | REVIEW |
| Control 4 | EML4–ALK | 80 | 0 | 1 | −2 / +2 | 58 / 19 | REVIEW |
| Control 5 | EML4–ALK | 80 | 0 | 1 | −2 / +2 | 59 / 20 | REVIEW |
| Control 6 | CD74–ROS1 | 78 | 0 | 1 | +2 / +2 | 54 / 13 | REVIEW |
| Control 7 | CD74–ROS1 | 75 | 0 | 1 | +2 / +2 | 52 / 11 | REVIEW |
| Total | 553 | 0 / 7 controls | 7 / 7 controls | ≤ 2 absolute | 385 / 121 | 0 PASS/HIGH | |
Concordance with the direct FASTQ mode
Relative to the earlier direct targeted-FASTQ analysis, target detection, reportability, breakpoint error, tier, score, and split/spanning support were identical in all seven controls. Whole-reference BAM ingestion enumerated fewer broad candidates: 31,565 to 1,024 across backgrounds and 31,572 to 1,031 across spiked datasets. The 97 background candidates sharing a target gene pair fell to zero. The two input modes enumerate background candidates differently, so the locked target endpoint and its associated call fields—not the broad candidate totals— are the directly concordant comparison. These changes are not an estimate of clinical specificity.
5.2 Alignment-field realism
The 553 planted pairs produced 1,106 primary and 417 secondary alignments. No planted primary
record was unmapped. With the historical BWA -M behavior, shorter split hits were
represented as secondary rather than supplementary alignments; this is expected aligner output.
| Measure | Observed | Interpretation |
|---|---|---|
| Mapped planted primary records | 1,106 / 1,106 (100%) | Every planted read end received a primary placement |
AS | 1,106 / 1,106 (100%) | Calculated by BWA from each reported alignment |
XS | 1,106 / 1,106 (100%) | Calculated from competing placements in these records; not copied from real reads |
NM, MD, and read group | 1,106 / 1,106 for each field | All required mapped-primary fields were present |
SA | 417 / 1,106 (37.7%) | Present where BWA emitted a linked chimeric/split representation; it is conditional, not universal |
| Secondary / supplementary records | 417 / 0 | Consistent with the selected BWA -M convention |
| Primary MAPQ | Median 60; range 36–60 | Placement confidence was calculated by the aligner |
| Clipped primary records | 544 / 1,106 (49.2%) | Clipping emerged from alignment rather than direct BAM construction |
In the earlier direct BAM audit, 0% of planted records carried AS or
XS. After FASTQ generation and BWA-MEM, 100% of mapped planted primary records
carried both in this experiment. More importantly, the values now agree with the same
alignment event that produced CIGAR, MAPQ, clipping, flags, mate fields, NM,
MD, and conditional SA.
5.3 Comparison with length-matched background reads
All 1,106 planted primary records were compared with 1,106 ordinary primary records sampled deterministically from the matched background BAMs. Background records were stratified by control, read end, and 10-base query-length band. This controls basic read-length composition; it does not make an ordinary genomic read biologically equivalent to a fusion-junction read.
| Measure | Planted fusion reads | Length-matched background |
|---|---|---|
| Records | 1,106 | 1,106 |
| Median MAPQ | 60 | 60 |
| Any clipping | 49.2% | 2.4% |
SA present | 37.7% | 0% |
| Proper-pair flag | 47.7% | 95.8% |
The planted reads are intentionally chimeric. Their higher clipping and SA rates,
and lower proper-pair rate, are expected manifestations of fusion structure. The comparison
shows aligner-generated behavior; it does not claim that synthetic reads are identical to
naturally occurring fusion molecules or ordinary background reads.
5.4 FusionSieve evidence
The seven target candidates contained 385 unique split reads and 121 unique spanning pairs,
for 506 unique supporting observations. Each candidate had score 1.0 and no hard filters.
Five candidates carried the additional soft filter low_depth; all seven carried
reporting-readiness blockers described below. The score is a ranking score, not a probability
of disease or a clinical confidence estimate.
5.5 Integrity checks
| Check | Result |
|---|---|
| Alignment manifests | 14 / 14 verified |
| FusionSieve rerun manifests and result JSON | 14 / 14 verified |
| Referenced file sizes and SHA-256 values | All verified |
| Matched backgrounds with zero target-window candidate | 7 / 7 |
| Private identifiers exported in aggregate results | None |
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What REVIEW and PASS/HIGH mean
The phrase “none reached PASS or HIGH” does not mean the planted fusions were missed. All seven were detected and retained as REVIEW candidates. The terms describe two different decisions:
| Question | Rule in this study | Observed result |
|---|---|---|
| Was the target detected? | One REVIEW, PASS, or HIGH candidate within the locked window, no hard filters, and zero matched-background window calls | Yes: 7 / 7 |
| Was it automatically reportable? | The candidate must be PASS or HIGH and the reporting-readiness gate must be satisfied | No: 0 / 7 |
All seven candidates were kept at REVIEW because automatic reporting was disabled, the heuristic scorer had not been independently validated, no compatible panel of normals was supplied, and duplicate-processing provenance could not be fully verified from reconstructed FASTQs. Controls 1–5 also had a soft low-depth flag at one side of the breakpoint. Controls 6–7 did not have that depth flag, but the same fail-closed readiness gate still applied.
Raising a REVIEW candidate to PASS/HIGH by editing a threshold or enabling a switch would not create the missing validation evidence. Honest automatic reporting requires a validated model, a compatible panel of normals, verified processing provenance, and a prospectively accepted reporting configuration.
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Conclusions
- The missing full-reference alignment step was completed successfully for all 14 matched datasets.
- FASTQ-level generation followed by BWA-MEM removed the need to invent
AS,XS, MAPQ, CIGAR, flags, or mate fields. - FusionSieve recovered the intended target-window candidate in all seven spiked BAMs and in none of the matched backgrounds.
- The aligner-generated records passed the reported tag, sorting, index, and file-integrity checks.
- The candidates remain REVIEW-tier research findings. This study does not support automatic reporting or any clinical claim.
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Limitations
- The original instrument FASTQs were unavailable. FASTQ reconstruction cannot restore discarded duplicate molecules, original headers, raw file order, or pre-processing quality values.
- Hard-clipped bases are absent from BAM records and cannot be reconstructed. This explains why some BAM-derived FASTQ query lengths are shorter than the original sequencer read length.
- The deterministic reorder removes coordinate clustering but cannot recreate the historical flow-cell order or exact BWA batch boundaries.
- This is a pinned BWA-MEM replay, not a byte-for-byte recreation of the unavailable historical Picard/GATK post-processing chain.
- The hg19 FASTA is SHA-256 locked and contig-compatible with the source headers, but the historical BAM headers did not contain sequence MD5 fields that would prove whole-reference identity retrospectively.
- Only seven backgrounds and two fusion definitions were evaluated. This is not a sensitivity, specificity, precision, reproducibility, or limit-of-detection study.
- FusionSieve automatic reporting remained intentionally fail-closed because scorer, panel-of-normals, and processing-provenance requirements were not met.
- No biological outcome, patient outcome, treatment response, or clinical decision was evaluated.
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Future directions
- Recover original instrument FASTQs, if available, and repeat the replay without reconstruction.
- Reproduce the full historical post-processing chain when the exact Picard/GATK resources and intermediates can be verified.
- Expand the fusion panel across more genes, breakpoint architectures, read lengths, doses, and background depths.
- Run a blinded recovery study with truth locked before analysis and an independent caller.
- Validate reportability separately using a grouped training/validation design, a compatible panel of normals, and prospectively locked thresholds.
- Measure reproducibility across operating systems and production alignment environments.
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Software and downloads
SpikeForge is the local research-control generator used to create the FASTQ-level fusion pairs. The public software site contains installation and use instructions. BAM and FASTQ files remain local because they are large and may contain sensitive sequencing information.
SpikeForge software
Download the research preview and validate it locally before use.
Full-reference follow-up package
Methods, aggregate results, alignment comparison, runtime and reference hashes, workflow scripts, tests, integrity manifest, and privacy statement. The 59,636-byte package excludes BAMs, FASTQs, indexes, read names, laboratory identifiers, local paths, runtime binaries, caller logs, and private manifests. Privacy scan: PASS with zero findings; archive CRC and inventory checks: PASS; packaged tests: 6 / 6 passed.
SHA-256: 4e0b3a377dacbff1c00070bfcb5f8ea1c9beef9ae935990e687f1dd4bd3d3d4f
Earlier FASTQ functional-study package
This existing package documents the preceding targeted-panel FASTQ study. It is retained so previously shared links continue to work. Use the full-reference package above for the current BWA-MEM replay and alignment-realism results.
SHA-256: 2161b446fcc7bbc1483ed37453a3fd4d9ba7625157edd3dad5b9c366313e9714
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