I’m trying to assemble small (20Mb), diploid fungal genome from MiSeq reads (~400bp after merging, 100x coverage). The tricky thing: it’s heterozygous. The divergence is ~4%, but there are hundreds (thousands?) loss of heterozygosity (LOH) regions, accounting in total to almost half of the genome…Do you know of any assembler (methodology) capable of handling with such data?
Since we never wrote on Hapsembler, now is a good opportunity to go over the algorithm and its applicability.
Most traditional genome assemblers assume that both chromosomes are identical. More the genome under consideration moves away from that assumption, more difficult it is to assemble it from NGS (or even Sanger) sequences using traditional tools. BGI tried to assemble insanely polymorphic Oyster genome using SOAPdenovo2 and was not successful. Then they chemically broke the chromosomes into small bacteria-sized pieces and assembled each piece separately. Those smaller pieces were joined computationally using SOAPdenovo2c scaffolding algorithm.
Someone writing an assembly program for polymorphic genomes starts with asking
- do we want to retrieve two separate chromosomes, or one single genome that is mosaic of two difference sequences? It is easy to choose more general solution, but then the question of sorting out phases is important. That is not an easy problem to solve along with a new assembly.
Moral of the story - it is very difficult to assemble a highly polymorphic genomes using traditional assembly programs, unless you know the code inside out and write loops around various built in assumptions.
Hapsembler works through three stages -
**Alignment module: **
The alignment module is used to compute pairwise sequence alignments between reads that obey certain criteria. This procedure employs an efficient kmer hashing technique to detect overlaps longer than a user defined length.
Error correction module:
This initial set of overlaps is used to correct sequencing errors by a probabilistic error correction procedure based on Naive Bayes. The corrected reads are then passed through another overlapping stage to compute the final set of overlaps.
The graph module builds an overlap graph (a.k.a string graph) using the
overlaps reported by the alignment module. This graph is used to construct
path sets representing possible tilings between paired reads. A mate pair graph
is then constructed in which nodes represent mate pairs, and edges represent
possible overlaps between mate pairs as constrained by the path sets. Finally,
contigs are determined by finding maximal paths in the mate pair graph.
You can find more details in their paper.
In this context, readers may also check HaploMerger: A Software Tool for Assembling Highly Polymorphic Genomes
On a somewhat related topic, readers may like to learn about Cortex assembler from Zamin Iqbal and read our earlier commentary titled Exploring Single-sample SNP and indel Calling with Whole-Genome Assembly. If you really looking for an esoteric graph-based algorithm for sorting out phases, please try HapCompass from Sorin Istrail’s lab.