Computational protocol: The loss of photosynthetic pathways in the plastid and nuclear genomes of the non-photosynthetic mycoheterotrophic eudicot Monotropa hypopitys

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Protocol publication

[…] A single Monotropa hypopitys plant was collected from the Vologda region, Russia (58°35’14” N, 37°59’20” E). The specimen is stored under accession number MON-1VOLR. Total genomic DNA was extracted from fresh leaves of a single individual and sequenced with a Roche Genome Sequencer GS FLX (Roche, Switzerland) using the Titanium XL+ protocol for a shotgun genome library. About 86 Mb of sequences with an average read length of 356 bp were generated. De novo assembly was performed with Newbler Assembler v. 2.9 (454 Life Sciences, Branford, USA), which yielded three chloroplast DNA contigs with 18-fold coverage. These contigs were identified based on sequence similarity to chloroplast genomes of related species (Ericales) and high coverage. The complete plastid genome sequence was obtained upon the generation of appropriate PCR fragments covering the gaps between the contigs and their sequencing by Sanger method on ABI PRISM 3730 (Applied Biosystems). To verify the correct assembly of the reconstructed plastid genome, raw reads were mapped against the reconstructed sequence with GS Reference mapper (454 Life Sciences, Branford, USA). Plastid genome annotation was performed using DOGMA [] with further manual correction. Circular map of the plastome was drawn using OrganellarGenomeDRAW tools [].The sequence of the plastid genome of M. hypopitys MON-1VOLR was submitted to GenBank under accession number KU640957. [...] Leaves (flower bracts) and flowers of two individual Monotropa hypopitys plants (not the same specimen used for chloroplast genome sequencing) and two pooled samples of roots with haustoria were collected for transcriptome analysis. Total RNA was extracted from ~0.3 g tissue for each six samples using the RNeasy Plant Mini kit (Qiagen, Valencia, CA). RNA samples were sequenced using the Illumina HiSeq2500 platform (100-bp reads) according to the manufacturer’s instructions (Illumina Inc., USA). RNA-seq read data has been deposited in the NCBI SRA database under accession SRP069226. M. hypopitys transcriptome sequencing resulted in a total of 103 million high quality sequencing reads after primer and quality trimming with Cutadapt [] and Sickle (https://github.com/najoshi/sickle), respectively. Assembly of the transcriptome from the combined six RNA-seq datasets was carried out using the Trinity platform []. The transcriptome was assembled into 98350 unigenes ranging from 201 to 12993 bp in length with N50 of 1342 bp. Coding region identification in Trinity assembly was done using TransDecoder (http://transdecoder.github.io). Trinotate (https://trinotate.github.io/) was used to assign hits from TrEMBL and Swiss-Prot databases (http://www.uniprot.org/uniprot/), and to assign GO terms and pfam domains (accessed 15/02/2016). 37977 unigenes were annotated in the TrEMBL protein database using predicted protein sequences and 38419 unigenes were annotated in the Swiss-Prot database using BLASTX). Protein-coding genes were assigned with the KEGG orthology identifiers using web-based KAAS server [].The levels of transcription of protein-coding genes in the plastid genome were quantitated employing RSEM [] and Bowtie 2 program []. Transcription levels were expressed as fragments per kilobase of exon per million fragments mapped (FPKM) values. […]

Pipeline specifications

Software tools Newbler, DOGMA, OGDRAW, cutadapt, Trinity, TransDecoder, Trinotate, BLASTX, KAAS, RSEM, Bowtie
Databases SRA Pfam UniProt KO
Applications Genome annotation, RNA-seq analysis, Transcription analysis, Genome data visualization
Organisms Monotropa hypopitys
Chemicals Adenosine Triphosphate, Chlorophyll, NAD