In bioinformatics, sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology might not be able to 'read' whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically, the short fragments (reads) result from shotgun sequencing genomic DNA, or gene transcript (ESTs).

The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces. Besides the obvious difficulty of this task, there are some extra practical issues: the original may have many repeated paragraphs, and some shreds may be modified during shredding to have typos. Excerpts from another book may also be added in, and some shreds may be completely unrecognizable.

There are three approaches to assembling sequencing data:

Referenced-guided assembly is a combination of the other types. This type is applied on long reads to mimic short reads advantages (i.e. call quality). The logic behind it is to group the reads by smaller windows within the reference. Reads in each group will then be reduced in size using the k-mere approach to select the highest quality and most probable contiguous (contig). Contigs will then will be joined together to create a scaffold. The final consense is made by closing any gaps in the scaffold.

The first sequence assemblers began to appear in the late 1980s and early 1990s as variants of simpler sequence alignment programs to piece together vast quantities of fragments generated by automated sequencing instruments called DNA sequencers. As the sequenced organisms grew in size and complexity (from small viruses over plasmids to bacteria and finally eukaryotes), the assembly programs used in these genome projects needed increasingly sophisticated strategies to handle:

Faced with the challenge of assembling the first larger eukaryotic genomes—the fruit fly Drosophila melanogaster in 2000 and the human genome just a year later,—scientists developed assemblers like Celera Assembler and Arachne able to handle genomes of 130 million (e.g., the fruit fly D. melanogaster) to 3 billion (e.g., the human genome) base pairs. Subsequent to these efforts, several other groups, mostly at the major genome sequencing centers, built large-scale assemblers, and an open source effort known as AMOS was launched to bring together all the innovations in genome assembly technology under the open source framework.

Expressed sequence tag or EST assembly was an early strategy, dating from the mid-1990s to the mid-2000s, to assemble individual genes rather than whole genomes. The problem differs from genome assembly in several ways. The input sequences for EST assembly are fragments of the transcribed mRNA of a cell and represent only a subset of the whole genome. A number of algorithmical problems differ between genome and EST assembly. For instance, genomes often have large amounts of repetitive sequences, concentrated in the intergenic regions. Transcribed genes contain many fewer repeats, making assembly somewhat easier. On the other hand, some genes are expressed (transcribed) in very high numbers (e.g., housekeeping genes), which means that unlike whole-genome shotgun sequencing, the reads are not uniformly sampled across the genome.

EST assembly is made much more complicated by features like (cis-) alternative splicing, trans-splicing, single-nucleotide polymorphism, and post-transcriptional modification. Beginning in 2008 when RNA-Seq was invented, EST sequencing was replaced by this far more efficient technology, described under de novo transcriptome assembly.