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All NGS questions

Question 8 of 9 Intermediate Scenario

A sequencing run has excellent yield but poor mapping rates. How would you investigate?

Short answer

Separate sequencing output quality from biological and mapping utility. Excellent yield can coexist with wrong library identity, contamination, poor trimming or an inappropriate reference.

Why

Read yield says how many bases were emitted. Mapping rate depends on whether those bases resemble the chosen reference under the aligner settings. rRNA, adapters, low complexity, host contamination, mixed species and structural variation can all reduce alignments.

How to work it through

  1. Compare per-cycle quality, adapter content, insert size, duplication and read length across samples.
  2. Review read-level taxonomic composition or align a subset to plausible alternate references.
  3. Verify sample identity, reference version, strandedness, contamination, library type and correct paired-end processing.
  4. Re-evaluate aligner parameters only after ruling out wrong inputs, and preserve raw FASTQ and pipeline versions.

An example

An RNA-seq library with high base quality but mostly rRNA reads will produce strong instrument metrics and poor useful transcriptome mapping. Another sample may map poorly simply because the reference is the wrong organism or genome build.

Where the answer stops holding

Some true reads from novel organisms or highly rearranged tumors cannot map uniquely. Host depletion and metagenomics require different success metrics from germline WGS. A high mapping percentage can also hide severe reference bias.

A strong answer covers

  • Separate sequencing output quality from biological and mapping utility
  • Excellent yield can coexist with wrong library identity, contamination, poor trimming or an inappropriate reference
  • Compare per-cycle quality, adapter content, insert size, duplication and read length across samples

Common mistakes

  • Repeating sequencing before examining read content
  • Changing aligner flags until a metric improves without checking biological validity