text: In order to link sequence read abundance to the expression of a particular gene, transcript sequences are aligned to a reference genome or de novo aligned to one another if no reference is available. The key challenges for alignment software include sufficient speed to permit billions of short sequences to be aligned in a meaningful timeframe, flexibility to recognise and deal with intron splicing of eukaryotic mRNA, and correct assignment of reads that map to multiple locations. Software advances have greatly addressed these issues, and increases in sequencing read length reduce the chance of ambiguous read alignments. A list of currently available high-throughput sequence aligners is maintained by the EBI.
  Alignment of primary transcript mRNA sequences derived from eukaryotes to a reference genome requires specialised handling of intron sequences, which are absent from mature mRNA. Short read aligners perform an additional round of alignments specifically designed to identify splice junctions, informed by canonical splice site sequences and known intron splice site information. Identification of intron splice junctions prevents reads from being misaligned across splice junctions or erroneously discarded, allowing more reads to be aligned to the reference genome and improving the accuracy of gene expression estimates. Since gene regulation may occur at the mRNA isoform level, splice-aware alignments also permit detection of isoform abundance changes that would otherwise be lost in a bulked analysis.
  De novo assembly can be used to align reads to one another to construct full-length transcript sequences without use of a reference genome. Challenges particular to de novo assembly include larger computational requirements compared to a reference-based transcriptome, additional validation of gene variants or fragments, and additional annotation of assembled transcripts. The first metrics used to describe transcriptome assemblies, such as N50, have been shown to be misleading and improved evaluation methods are now available. Annotation-based metrics are better assessments of assembly completeness, such as contig reciprocal best hit count. Once assembled de novo, the assembly can be used as a reference for subsequent sequence alignment methods and quantitative gene expression analysis.