Software we have made:
Supported by the B2B Consortium Grant
MMAPPR (Mutation Mapping Analysis Pipeline for Pooled RNA-seq) is an analysis pipeline for mapping mutations using RNA-seq. It works without parental strain information, without the requirement of a pre-existing snp map of the organism, and without erroneous assumptions that recombination occurs at the same frequency across the genome. In addition, MMAPPR compensates for the considerable amount of noise in RNA-seq datasets and simultaneously identifies the region where the mutation lies and generates a list of putative coding region mutations in the linked genomic segment. MMAPPR can utilize RNA-seq datasets from isolated tissues or whole organisms that are often generated for phenotypic analysis and gene network analysis in novel mutants. Thus, MMAPPR is rapid, cost-efficient, and highly automated online pipeline that effectively puts the ability to perform gene mapping in every laboratory, studying any organism with a well assembled genome.
Hill, J. T., Demarest, B. L., Bisgrove, B. W., Gorsi, B., Su, Y.-C., & Yost, H. J. (2013). MMAPPR: Mutation Mapping Analysis Pipeline for Pooled RNA-seq. Genome Research. PMID: 23299975
Current Version: 0.83 (Last updated on 31 Jan 2013)Current Version
Poly Peak Parser
Background: Genome editing techniques, including ZFN, TALEN and CRISPR, have created a need to rapidly screen many F1 individuals to identify carriers of short indels and determine the sequences of the mutations. Current techniques require multiple clones of the targeted region to be sequenced for each individual, which is inefficient when many individuals must be analyzed. Direct Sanger sequencing of a PCR amplified region surrounding the target site is efficient, but Sanger sequencing genomes heterozygous for an indel results in a string of
double peaks due to the mismatched region.
Results: In order to facilitate indel identification, we developed an online tool called Poly Peak Parser that is able to separate chromatogram data containing ambiguous base calls into wild-type and mutant allele sequences, revealing the nature of the indel from a single sequencing run per individual performed directly on a PCR product spanning the targeted site, without cloning.
Conclusions: The method and algorithm described here facilitate rapid identification and sequence characterization of heterozygous mutant carriers generated by genome editing. Although designed for screening F1 individuals after genome editing, this tool can also be used to identify heterozygous indels in many contexts.
Hill JT, Demarest BL, Bisgrove BW, Su YC, Smith M, Yost HJ. (2014) Poly Peak Parser: Method and software for identification of unknown indels using Sanger Sequencing of PCR products. Developmental Dynamics. PMID: 25160973
The code used to make PolyPeakParser is available as the "sangerseqR" bioconductor package:
The sangerseqR package has been used to create a tool to estimate the frequency of somatic indels in TALEN or CRISPR injected animals or cell lines:
Users looking for a reference-free method should look into Indelligent:
Founder Principle-Driven Enrichment Calculator
Founder principle-driven enrichment (FPE) is a method that allows isolation of rare BAC recombinants (as rare as 1:10,000 to 1:100,000) without using selectable markers. The web-based calculator presented here enables users to easily determine all possible optimal FPE parameters for any desired cost function, i.e. the specific working conditions.
George T. Lyozin, Paul C. Bressloff, Amit Kumar, Yasuhiro Kosaka, Bradley L. Demarest, H. Joseph Yost, Michael R. Kuehn, and Luca Brunelli (2014) Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis. Nature Methods. PMID: 25028895