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Open Access Highly Accessed Open Badges Methodology article

A new method to compute K-mer frequencies and its application to annotate large repetitive plant genomes

Stefan Kurtz1, Apurva Narechania23, Joshua C Stein2 and Doreen Ware2*

  • * Corresponding author: Doreen Ware

  • † Equal contributors

Author Affiliations

1 Center for Bioinformatics, University of Hamburg, Bundesstraße 43, 20146 Hamburg, Germany

2 Cold Spring Harbor Lab, 1 Bungtown Rd, Williams #5, Cold Spring Harbor, NY 11724, USA

3 Sackler Institute for Comparative Genomics, American Museum of Natural History, New York, NY, USA

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BMC Genomics 2008, 9:517  doi:10.1186/1471-2164-9-517

Published: 31 October 2008



The challenges of accurate gene prediction and enumeration are further aggravated in large genomes that contain highly repetitive transposable elements (TEs). Yet TEs play a substantial role in genome evolution and are themselves an important subject of study. Repeat annotation, based on counting occurrences of k-mers, has been previously used to distinguish TEs from low-copy genic regions; but currently available software solutions are impractical due to high memory requirements or specialization for specific user-tasks.


Here we introduce the Tallymer software, a flexible and memory-efficient collection of programs for k-mer counting and indexing of large sequence sets. Unlike previous methods, Tallymer is based on enhanced suffix arrays. This gives a much larger flexibility concerning the choice of the k-mer size. Tallymer can process large data sizes of several billion bases. We used it in a variety of applications to study the genomes of maize and other plant species. In particular, Tallymer was used to index a set of whole genome shotgun sequences from maize (B73) (total size 109 bp.). We analyzed k-mer frequencies for a wide range of k. At this low genome coverage (≈ 0.45×) highly repetitive 20-mers constituted 44% of the genome but represented only 1% of all possible k-mers. Similar low-complexity was seen in the repeat fractions of sorghum and rice. When applying our method to other maize data sets, High-C0t derived sequences showed the greatest enrichment for low-copy sequences. Among annotated TEs, the most highly repetitive were of the Ty3/gypsy class of retrotransposons, followed by the Ty1/copia class, and DNA transposons. Among expressed sequence tags (EST), a notable fraction contained high-copy k-mers, suggesting that transposons are still active in maize. Retrotransposons in Mo17 and McC cultivars were readily detected using the B73 20-mer frequency index, indicating their conservation despite extensive rearrangement across cultivars. Among one hundred annotated bacterial artificial chromosomes (BACs), k-mer frequency could be used to detect transposon-encoded genes with 92% sensitivity, compared to 96% using alignment-based repeat masking, while both methods showed 92% specificity.


The Tallymer software was effective in a variety of applications to aid genome annotation in maize, despite limitations imposed by the relatively low coverage of sequence available. For more information on the software, see webcite.