TY - JOUR
T1 - MAPINS, a highly efficient detection method that identifies insertional mutations and complex DNA rearrangements
AU - Lin, Huawen
AU - Cliften, Paul F.
AU - Dutcher, Susan K.
N1 - Funding Information:
1This work was supported by grants from the Children’s Discovery Institute (PD-II-2014-379) and the National Institute of General Medical Sciences (R01 GM32843) to S.K.D. The Genome Technology Access Center (GTAC) in the Department of Genetics at Washington University School of Medicine performed whole sequencing, and it is partially supported by National Cancer Institute Cancer Center Support grant #P30 CA91842 to the Siteman Cancer Center and by the Institute for Clinical and Translational Sciences (ICTS)/Clinical and Translational Science Award (CTSA) grant UL1RR024992 from the National Center for Research Resources (NCRR), a component of the NIH and NIH Roadmap for Medical Research.We thank Mihaela Stoyanova and John Garza for technical support and Dr. Mathieu Bottier, Gervette M. Penny, and Manishi Pandey for comments on the article. Special thanks to Dr. Gary Stormo for the name of MAPINS. We thank one of the reviewers who suggested that we study the frequency of chimeric reads that are not linked to the selectable marker.
Funding Information:
1This work was supported by grants from the Children’s Discovery Institute (PD-II-2014-379) and the National Institute of General Medical Sciences (R01 GM32843) to S.K.D. The Genome Technology Access Center (GTAC) in the Department of Genetics at Washington University School of Medicine performed whole sequencing, and it is partially supported by National Cancer Institute Cancer Center Support grant #P30 CA91842 to the Siteman Cancer Center and by the Institute for Clinical and Translational Sciences (ICTS)/Clinical and Translational Science Award (CTSA) grant UL1RR024992 from the National Center for Research Resources (NCRR), a component of the NIH and NIH Roadmap for Medical Research. 2Author for contact: dutcher@wustl.edu. 3Senior author.
Publisher Copyright:
© 2018 American Society of Plant Biologists. All rights reserved.
PY - 2018/12/1
Y1 - 2018/12/1
N2 - Insertional mutagenesis, in which a piece of exogenous DNA is integrated randomly into the genomic DNA of the recipient cell, is a useful method to generate new mutants with phenotypes of interest. The unicellular green alga Chlamydomonas reinhardtii is an outstanding model for studying many biological processes. We developed a new computational algorithm, MAPINS (map-ping insertions), to efficiently identify insertion sites created by the integration of an APHVIII (aminoglycoside 3′-phosphotransferase VIII) cassette that confers paromomycin resistance. Using whole-genome sequencing data, this method eliminates the need for genomic DNA manipulation and retains all the sequencing information provided by paired-end sequencing. We experimentally verified 38 insertion sites out of 41 sites (93%) identified by MAPINS from 20 paromomycin-resistant strains. Using meiotic analysis of 18 of these strains, we identified insertion sites that completely cosegregate with paromomycin resistance. In six of the seven strains with a mutant phenotype, we demonstrated complete cosegregation of the mutant phenotype and the verified insertion site. In addition, we provide direct evidence of complex rearrangements of genomic DNA in five strains, three of which involve the APHVIII insertion site. We suggest that strains obtained by insertional mutagenesis are more complicated than expected from previous analyses in Chlamydomonas. To map the locations of some complex insertions, we designed 49 molecular markers based on differences identified via whole-genome sequencing between wild-type strains CC-124 and CC-125. Overall, MAPINS provides a low-cost, efficient method to characterize insertional mutants in Chlamydomonas.
AB - Insertional mutagenesis, in which a piece of exogenous DNA is integrated randomly into the genomic DNA of the recipient cell, is a useful method to generate new mutants with phenotypes of interest. The unicellular green alga Chlamydomonas reinhardtii is an outstanding model for studying many biological processes. We developed a new computational algorithm, MAPINS (map-ping insertions), to efficiently identify insertion sites created by the integration of an APHVIII (aminoglycoside 3′-phosphotransferase VIII) cassette that confers paromomycin resistance. Using whole-genome sequencing data, this method eliminates the need for genomic DNA manipulation and retains all the sequencing information provided by paired-end sequencing. We experimentally verified 38 insertion sites out of 41 sites (93%) identified by MAPINS from 20 paromomycin-resistant strains. Using meiotic analysis of 18 of these strains, we identified insertion sites that completely cosegregate with paromomycin resistance. In six of the seven strains with a mutant phenotype, we demonstrated complete cosegregation of the mutant phenotype and the verified insertion site. In addition, we provide direct evidence of complex rearrangements of genomic DNA in five strains, three of which involve the APHVIII insertion site. We suggest that strains obtained by insertional mutagenesis are more complicated than expected from previous analyses in Chlamydomonas. To map the locations of some complex insertions, we designed 49 molecular markers based on differences identified via whole-genome sequencing between wild-type strains CC-124 and CC-125. Overall, MAPINS provides a low-cost, efficient method to characterize insertional mutants in Chlamydomonas.
UR - http://www.scopus.com/inward/record.url?scp=85058578199&partnerID=8YFLogxK
U2 - 10.1104/pp.18.00474
DO - 10.1104/pp.18.00474
M3 - Article
C2 - 30206105
AN - SCOPUS:85058578199
VL - 178
SP - 1436
EP - 1447
JO - Plant Physiology
JF - Plant Physiology
SN - 0032-0889
IS - 4
ER -