De novo assembly of human genomes with massively parallel short read sequencing

Abstract

Next-generation massively parallel DNA sequencing technologies provide ultrahigh throughput at a substantially lower unit data cost; however, the data are very short read length sequences, making de novo assembly extremely challenging. Here, we describe a novel method for de novo assembly of large genomes from short read sequences. We successfully assembled both the Asian and African human genome sequences, achieving an N50 contig size of 7.4 and 5.9 kilobases (kb) and scaffold of 446.3 and 61.9 kb, respectively. The development of this de novo short read assembly method creates new opportunities for building reference sequences and carrying out accurate analyses of unexplored genomes in a cost-effective way.

Figure 1.

(A) Length distribution of unique and repeat sequence clusters in the human genome. At each chromosomal location, we checked the frequency of the 25-mer in the whole human genome. If it appeared once, we defined it as unique; otherwise it was considered a repeat 25-mer. The regions were then merged as unique clusters and repeat clusters, and those small unique clusters (<100 bp) inside repeat clusters were defined as repeats. (B) Sequence length distribution of an ideal assembly with each insert-sized paired-ends. The repeat clusters with lengths smaller than the assumed insert size of paired-ends were crossed and the unique clusters were merged. These unique clusters represent the ideal assembly using the paired-ends.

Figure 2.

Schematic overview of the assembly algorithm. (A) Genomic DNA was fragmented randomly and sequenced using paired-end technology. Short clones with sizes between 150 and 500 bp were amplified and sequenced directly; while long range (2–10 kb) paired-end libraries were constructed by circularizing DNA, fragmentation, and then purifying fragments with sizes in the range of 400–600 bp for cluster formation. (B) The raw or precorrected reads were then loaded into computer memory and de Bruijn graph data structure was used to represent the overlap among the reads. (C) The graph was simplified by removing erroneous connections (in red color on the graph) and solving tiny repeats by read path: (i) Clipping the short tips, (ii) removing low-coverage links, (iii) solving tiny repeats by read path, and (iv) merging the bubbles that were caused by repeats or heterozygotes of diploid chromosomes. (D) On the simplified graph, we broke the connections at repeat boundaries and output the unambiguous sequence fragments as contigs. (E) We realigned the reads onto the contigs and used the paired-end information to join the unique contigs into scaffolds. (F) Finally, we filled in the intrascaffold gaps, which were most likely comprised by repeats, using the paired-end extracted reads.

Figure 3.

Examples of deletion and insertion identified in the comparison of the assembled individual human genomes and the NCBI reference genome. (A) A 17-bp deletion in scaffold27122121 of the African genome located on chromosome 7. (B) A 7926-bp insertion in scaffold4928 of the Asian genome located on chromosome 7. The inserted sequence fragment was validated by a human BAC clone AC153461.2 in GenBank, and also exists in the chimpanzee genome.

Figure 4.

N50 and N90 size of assembled contigs by different sequence depths. We sampled subsets of randomly selected reads from the Asian genome data for de novo assembly of contigs. The same K-mer (K = 25) size was used for all the assemblies.