Computational protocol: Sequencing and Analysis of Plastid Genome in Mycoheterotrophic Orchid Neottia nidus-avis

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Protocol publication

[…] Total DNA was extracted from the above-ground part of three Neottia plants using NucleoSpin Plant II kit (Macherey-Nagel, Germany). DNA (10 μg) was used for sequencing with Roche Genome Sequencer FLX system using the Titanium kit (454 Life Sciences). Sequencing was performed at the University of Illinois at Urbana-Champaign, W.M. Keck Center for Comparative and Functional Genomics. The sample was run on a half of a picotiter plate. The sequencing resulted in 590640 reads with an average read length of 528 bp. The output from the sequencing system in Standard Flowgram Format was converted to FASTA. Then a BLAST database was made from the resulting FASTA file and a plastome of Oncidium, the photosynthetic orchid was queried against it. All reads with e-value lower then 10−10 were used for de novo assembly using MIRA assembler ver. 3.2.0 (). The assembly resulted in 23 contigs with length more than 1,000 nucleotides. Two largest contigs (11,141 and 49,963 nucleotides) were used as a basis for the generation of draft version of the Neottia plastome. They were aligned with Oncidium plastome using the whole VISTA genome alignment tool (). Then Sanger sequencing was used to fill the gaps. To check the accuracy of the assembly and to correct possible 454 sequencing errors associated with homopolymer runs, several regions were sequenced by Sanger sequencing using the primers designed on the base of the assembly (supplementary table S1, Supplementary Material online). Average coverage for regions derived from 454 assembly is assessed as 29.8×; for regions sequenced by Sanger sequencing it is 2×. Polymerase chain reaction (PCR) amplification was performed on Biometra T300 thermal cycler using Encyclo PCR kit (Evrogen, Russia). PCR conditions were as follows: initial denaturation 3 min at 94 °C, then 35 cycles of 15 s at 94 °C, 25 s at 59 °C, and 1–5 min (depending on the expected length of the product) at 72 °C. Sanger sequencing was performed in the interinstitutional sequencing center at Engelhardt Institute of Molecular Biology (Moscow, Russia) using ABI PRISM BigDye Terminator kit v. 3.1 with following analysis on ABI PRISM 3730 genetic analyzer (Applied Biosystems). Initial annotation was produced using DOGMA (). Then manual correction and adjustment, that included alignment of every Oncidium and Phalaenopsis gene with Neottia plastome sequence was performed. The regions with similarity to known protein-coding genes but lacking intact ORF were classified as pseudogenes. To detect tRNAs pseudogenes, each tRNA-like sequence was analyzed using tRNAscan-SE () and by comparison with its putative orthologs from Oncidium and Phalaenopsis. Those sequences that lack typical tRNA folding and/or differ from their orthologs by multiple indels or substitutions were considered to be pseudogenes. The map of Neottia plastome was visualized using OGDRAW online tool () with further manual correction. Assembled and corrected sequence of Neottia plastome was deposited in the GenBank under accession number JF325876.For dN/dS calculation, all protein-coding plastid genes that are shared between Neottia, Rhizanthella, Oncidium, and Phalaenopsis were included in the analysis. Alignment was performed using ClustalW (); dN/dS ratios were calculated using codeml program from the package PAML 4.3 (). In the alignments of several genes, nontriplet indels or in-frame stop codons were found near 3′-end. In these cases, only the part of sequence before presumable indel or stop codon was used for dN/dS calculation. […]

Pipeline specifications

Software tools DOGMA, tRNAscan-SE, OGDRAW, Clustal W, PAML
Applications Genome annotation, Nucleotide sequence alignment, Genome data visualization
Organisms Aneura mirabilis, Rhizanthella gardneri, Marchantia polymorpha, Neottia nidus-avis, Epifagus virginiana