CHILD: a new tool for detecting low-abundance insertions and deletions in standard sequence traces

Abstract

Several methods have been proposed for detecting insertion/deletions (indels) from chromatograms generated by Sanger sequencing. However, most such methods are unsuitable when the mutated and normal variants occur at unequal ratios, such as is expected to be the case in cancer, with organellar DNA or with alternatively spliced RNAs. In addition, the current methods do not provide robust estimates of the statistical confidence of their results, and the sensitivity of this approach has not been rigorously evaluated. Here, we present CHILD, a tool specifically designed for indel detection in mixtures where one variant is rare. CHILD makes use of standard sequence alignment statistics to evaluate the significance of the results. The sensitivity of CHILD was tested by sequencing controlled mixtures of deleted and undeleted plasmids at various ratios. Our results indicate that CHILD can identify deleted molecules present as just 5% of the mixture. Notably, the results were plasmid/primer-specific; for some primers and/or plasmids, the deleted molecule was only detected when it comprised 10% or more of the mixture. The false positive rate was estimated to be lower than 0.4%. CHILD was implemented as a user-oriented web site, providing a sensitive and experimentally validated method for the detection of rare indel-carrying molecules in common Sanger sequence reads.

Base-Calling Algorithm with Vocabulary (BCV) Method for Analyzing Population Sequencing Chromatograms

“…The ability of the BCV package to detect indels of different sizes in minor DNA variants, which were presented in mixtures in different portions, was estimated using two test datasets, which were provided with CHILD [27] software. Both datasets contained chromatograms for mixtures of two clones of human mtDNA fragments with one of them having a deletion.…”

“…More complicated is the identification of indels using direct sequencing data. Currently, some algorithms allow indel detection in heterozygous genomes[23]–[27].…”

“…The RipSeq software can detect the presence in the mixture of up to three species from a vocabulary, and it does not generate sequences at the output. Practical methods of indel detection in direct sequencing chromatograms without a known wild-type also exist [25], [27], [32]. The Indelligent tool [25] identifies the two most similar sequences containing deletions assuming heterozygota.…”

“…The Indelligent tool [25] identifies the two most similar sequences containing deletions assuming heterozygota. The CHILD [27] aligns the primary and secondary sequences that were extracted from a chromatogram by Phred; the alignment process uses SSEARCH [33]. CHILD can detect indels in low fractions (5–10%) of DNA mixture.…”

Biodiversity Measures in Agriculture Using DNA

“…The most efficient are oligonucleotide arrays (Gupta et al, 2008). Similarly, small insertion or deletion (INDEL) have been localized and their frequencies measured (Zhidkov et al, 2011). Those markers are much more abundant and precise, and should turn out to be the most used, at least among the most studied species.…”