Computational protocol: Identification of the agg1 mutation responsible for negative phototaxis in a “wild-type” strain of Chlamydomonas reinhardtii

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Protocol publication

[…] Cell walls were removed from cells of agg1 and AGG1 (selected from the progenies of an agg1× CC-125 cross) strains by autolysin treatment . DNA was prepared using a DNeasy Plant Mini kit (QIAGEN) following the manufacturer's instructions. Two micrograms of each DNA sample was fragmented using a Covaris sonicator. ~300 bp DNA fragments were then purified using a Pippin Prep system (Sage Science) and were used to construct sequence libraries using an Illumina TruSeq library prep kit (Illumina) essentially following the manufacturer's instruction except that the number of PCR cycles in the amplification enrichment step was reduced to six to minimize the PCR amplification bias. The libraries were sequenced using Illumina HiSeq 2000 to produce 2×101 bp paired-end reads. In total, 48.2 M (9.7 Gbp) and 71.6 M paired-end reads (14.5 Gbp) were obtained for agg1 and the AGG1 strain, respectively.Sequence reads were aligned onto the Joint Genome Institute (JGI) version 5.3.1 (Creinhardtii_236.fa.gz) Chlamydomonas genome sequence (https://phytozome.jgi.doe.gov/pz/portal.html#! info? alias=Org_Creinhardtii) using bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml). The resulting SAM (Sequence Alignment/Map) files were converted to BAM (Binary Sequence Alignment/Map) and sorted by SAMtools version 0.1.18 (http://samtools.sourceforge.net/). To identify the mutation in the mapped region of agg1 genome, the alignment data were visualized using IGV software (version 2.3.39; https://www.broadinstitute.org/igv/) and compared to each other or to the genome database. […]

Pipeline specifications

Software tools Bowtie2, SAMtools, IGV
Databases Phytozome
Application WGS analysis
Organisms Chlamydomonas reinhardtii