{"title": "Analysis Pipeline FAQ", "status": "open", "content": [{"file": "/docs/public/pipeline_faq.jsx", "status": "open", "options": {"filetype": "jsx", "collapsible": false, "default_open": true, "convert_ext_links": true, "initial_header_level": 2}, "consortia": [{"@type": ["Consortium", "Item"], "@id": "/consortia/358aed10-9b9d-4e26-ab84-4bd162da182b/", "display_title": "SMaHT", "uuid": "358aed10-9b9d-4e26-ab84-4bd162da182b", "status": "open", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}], "identifier": "pipeline_faq", "date_created": "2025-10-10T16:21:09.142999+00:00", "section_type": "Page Section", "submitted_by": {"error": "no view permissions"}, "last_modified": {"modified_by": {"error": "no view permissions"}, "date_modified": "2026-09-10T17:13:27.814068+00:00"}, "schema_version": "1", "submission_centers": [{"@type": ["SubmissionCenter", "Item"], "status": "open", "display_title": "HMS DAC", "@id": "/submission-centers/9626d82e-8110-4213-ac75-0a50adf890ff/", "uuid": "9626d82e-8110-4213-ac75-0a50adf890ff", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}], "@id": "/static-sections/6c096d6a-d9ff-43ec-b6f6-10ebf8e55c5d/", "@type": ["StaticSection", "UserContent", "Item"], "uuid": "6c096d6a-d9ff-43ec-b6f6-10ebf8e55c5d", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}, "display_title": "pipeline_faq", "content": "\n
\n

Frequently Asked Questions

\n\n
\n
\n What processing and alignment steps are applied by the SMaHT Data Analysis Center (DAC) to the originally submitted sequencing data in the FASTQ or unaligned BAM format?\n
\n Illumina paired-end\n
    \n
  • All reads - both mapped and unmapped - are retained. No read is discarded, and the whole reads are retained. No trimming (\u201chard clipping\u201d) of reads is performed.
    • \n
  • The only reads removed are those affected by polyG artifacts, which arise on one- and two-channel sequencers (e.g., NovaSeq) when the dark base G is incorrectly called after synthesis termination. These artifacts typically appear as long G-runs at the ends of reads and can accumulate in GC-rich regions (e.g., chr2:32916230-32916625), creating downstream alignment issues.
    • \n
  • Original quality scores are retained in the OQ tag, enabling regeneration of FASTQ files with the original base qualities.
    • \n
  • Read groups are updated to include SMaHT portal identifiers for SM and LB.
    • \n
\n
\n PacBio\n
    \n
  • All reads - both mapped and unmapped - are retained. No read is discarded, and the whole reads are retained. No trimming (\u201chard clipping\u201d) of reads is performed.
    • \n
  • Non-essential kinetic tags are removed to reduce file size, but methylation-related tags (MM, ML) and assay-specific tags (e.g., Fiber-seq) are preserved.
    • \n
  • Read groups are modified to include SMaHT portal identifiers for SM and LB.
    • \n
\n
\n ONT\n
    \n
  • All reads - both mapped and unmapped - are retained. No read is discarded, and the whole reads are retained. No trimming (\u201chard clipping\u201d) of reads is performed.
    • \n
  • Non-essential kinetic tags are removed to reduce file size, while methylation-related tags (MM, ML) and assay-specific tags are preserved.
    • \n
  • Read groups are updated to include SMaHT portal identifiers for SM and LB.
    • \n
\n
\n
\n\n
\n How are CRAM files generated?\n
\n CRAM files are generated from BAM using Samtools view.\n
\n
\n Command:\n
\n
\n samtools view -@ {``} -hC -T {``} -o {``} {``}\n
\n
\n
\n\n
\n Which reference genome is used for CRAM conversion?\n
\n CRAM files are generated using the standard reference genome agreed upon by the consortium.\n
\n
\n The specific version in use is GCA_000001405.15 no_alt_analysis_set, accessible for download here in the following file: GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.gz\n
\n
\n This version excludes ALT contigs and Human decoy sequences from hs38d1 (GCA_000786075.2), and includes the following sequences:\n
    \n
  • Chromosomes from the GRCh38 Primary Assembly unit.
    • \n
  • Mitochondrial genome from the GRCh38 non-nuclear assembly unit.
    • \n
  • Unlocalized scaffolds from the GRCh38 Primary Assembly unit.
    • \n
  • Unplaced scaffolds from the GRCh38 Primary Assembly unit.
    • \n
  • Epstein-Barr virus (EBV) sequence.
    • \n
\n
\n Note: The two PAR regions on chrY have been hard-masked with Ns, and the chromosome Y sequence is not identical to the GenBank sequence but shares the same coordinates. Similarly, duplicate copies of centromeric arrays and WGS on chromosomes 5, 14, 19, 21 & 22 have been hard-masked with Ns.\n
\n
\n Note: The EBV sequence is not part of the genome assembly but is included in the analysis set for aligning reads often present in sequencing samples.\n
\n
\n\n
\n How can I convert a CRAM file back to BAM? \n
\n You can convert any CRAM file back to BAM using Samtools view.\n
\n
\n Command:\n
\n
samtools view -@ {``} -hb -T {``} {``} {`>`} {``}
\n
\n
    \n
  • view decodes the CRAM file with the reference (-T) and produces BAM (-b) with header (-h).
    • \n
\n
\n
\n\n
\n How can I generate an unaligned BAM (uBAM) from a CRAM file? \n
\n You can use Picard RevertSam. This command removes all alignment information, restores original qualities from the OQ tag if present, and sanitizes the file to enforce mate consistency (if paired-end data) while discarding non-primary alignments.\n
\n
\n Command:\n
\n
picard RevertSam \\
I={``} \\
O={``}
{`\\ REFERENCE_SEQUENCE=`}{``} \\
REMOVE_ALIGNMENT_INFORMATION=true
RESTORE_ORIGINAL_QUALITIES=true
SANITIZE=true
KEEP_FIRST_DUPLICATE=true
\n
\n
\n Use the -Xmx argument to set Java heap space if needed (e.g., -Xmx32g allocates 32G).\n
\n
\n \n
\n How can I regenerate FASTQ files from a CRAM file?\n
\n Illumina paired-end\n
\n Use Samtools view, Samtools collate, and Samtools fastq in combination. Both mapped and unmapped reads are retained.\n
\n
\n Command:\n
\n
samtools view -@ {``} -hb -T {``} {` `} {`| `}\\
{`samtools collate -@`} {``} {`-f -r 10000000 -u -O - | `}\\
{`samtools fastq`} \\
\n
\n -1 {``}{`.1.fastq.gz`} \\
{`-2 `}{``}{`.2.fastq.gz`} \\
{`-0 /dev/null -s /dev/null -n`} \\
-O \\
-@ {``} -\n
\n
\n
\n
    \n
  • view decodes the CRAM file with the reference (-T) and produces BAM (-b) with header (-h).
    • \n
  • collate ensures read pairs are sorted correctly. Fast mode (-f) uses an in-memory buffer (-r) to speed up pairing (10M alignments ~12GB RAM).
    • \n
  • fastq writes paired reads to {``}.1.fastq.gz and {``}.2.fastq.gz, one mate per file. -O uses OQ quality scores if available. Unpaired and singleton reads are discarded (-0 /dev/null -s /dev/null) - this is actually not necessary for DAC generated data since we do not discard individual mates during alignment and we don't have singleton or unpaired reads in the CRAM file; added to the command for completion.
    • \n
\n\n
\n PacBio\n
\n Use Samtools view and Samtools fastq in combination. Both mapped and unmapped reads are retained.\n
\n
\n Command:\n
\n
\n samtools view -@ {``} -hb -T {``} {``} | \\
samtools fastq -n -@ {``} - | \\
bgzip -@ {``} {`>`} {``}.fastq.gz\n
\n
    \n
  • view decodes the CRAM file with the reference (-T) and produces BAM (-b) with header (-h).
    • \n
  • fastq / bgzip writes all reads to {``}.fastq.gz. -n preserves original read names.
    • \n
\n
\n Note: FASTQ cannot carry SAM auxiliary tags (e.g., MM/ML tags for methylation). If converted to FASTQ, these tags are lost unless re-annotated after re-alignment.\n \n
\n
\n Re-alignment using pbmm2\n
\n If re-aligning using pbmm2 (https://github.com/PacificBiosciences/pbmm2), you can simply generate the aligned BAM from the CRAM file and run pbmm2 directly. pbmm2 will ignore non-primary alignments and import all tags correctly.\n\n
\n
\n ONT\n
\n Use Samtools view and Samtools fastq in combination. Both mapped and unmapped reads are retained.\n
\n
\n Command:\n
\n
samtools view -@ {``} -hb -T {` `} | \\
samtools fastq -n -@ {``} - | \\
bgzip -@ {``} {`>`} {``}.fastq.gz
\n
\n
    \n
  • view decodes the CRAM file with the reference (-T) and produces BAM (-b) with header (-h).
    • \n
  • fastq / bgzip writes all reads to {``}.fastq.gz. -n preserves original read names.
    • \n
\n\n
\n Note: FASTQ cannot carry SAM auxiliary tags (e.g., MM/ML tags for methylation). If converted to FASTQ, these tags are lost unless re-annotated after re-alignment.\n\n
\n
\n Re-alignment using minimap2\n
\n If re-aligning using minimap2, FASTQ can be streamed directly into minimap2 while carrying over the desired tags.\n
\n
\n Command:\n
\n
samtools view -@ {``} -hb -T {` `} | \\
samtools fastq -n -@ {``} -T {`,`} - |
minimap2 -y {`<...>`} {`>`} {``}
\n
\n
    \n
  • view decodes the CRAM file with the reference (-T) and produces BAM (-b) with header (-h).
    • \n
  • fastq extracts all reads while preserving read names (-n) and appending the specified tags (-T). In minimap2, the -y option re-imports these annotated tags into the alignment.
    • \n
\n
\n
\n\n
\n
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