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Sample GSM2252320

Query DataSets for GSM2252320

Status

Public on Feb 16, 2018

Title

Désirée_mock3

Sample type

SRA

Source name

control-mock inoculated potato plants

Organism

Solanum tuberosum

Characteristics

cultivar: Desiree
tissue: leaf
treatment: control
genotype: Desiree

Treatment protocol

After 4 weeks of growth in soil, the potato plants were inoculated with the sap of PVYNTN-infected plants (isolate NIB-NTN, accession no. AJ585342). For the mock inoculated plants, the same procedures were performed with the sap of healthy potato plants. Three plants for each treatment were used. PVYNTN- and mock-inoculated leaves of each genotype (Désirée, NahG-Désirée) were collected three days post inoculation (3 dpi) and immediately frozen in liquid nitrogen.

Growth protocol

Plants were grown in a growth chamber and kept at 21 C in the light and 18 C in the dark, at a relative humidity of 75%, with 70–90 mmol/m2/s2 radiation and a 16-h photoperiod.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from homogenised leaf tissue using TRIzol reagent (Invitrogen).
small RNA libraries were prepared for sequencing using standard Illumina protocols at Seqmatic.

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2000

Description

small RNAs

Data processing

Sequenced reads were trimmed for adaptor sequence. Low complexity reads containing at most two distinct nucleotides were removed.
For identification of known and novel miRNAs reads shorter than 18 nt and longer than 25 nt long, reads without adaptor, reads matching tRNA/rRNA sequences in RFAM version 10, the Genomic tRNA Database and EMBL, release 95, and reads not mapped to Solanum tuberosum genome (PGSC_DM_v4.3) using the filter tool from UEA sRNA Toolkit. The remaining reads were compared to all known miRNAs registered in miRBase database release 21, allowing no mismatches. The sequences that perfectly matched with mature miRNAs from other plants than potato, were mapped to the potato genome in order to identify MIR loci. For all identified MIR loci the pre-miRNA sequence was extracted. Some of these miRNA sequences were considered as miRNA variants/isomiRs and numbered by decreasing read number. To identify novel unannotated miRNAs, filtered reads were submitted to miRCat tool of the UEA sRNA Toolkit using suggested parameters for plants with minimum size of 18 and maximum size of 24 nt. Reads were first mapped to the genome, then the 100 and 200 nt long windows around the aligned reads were extracted and folded using RNAfold. The novelty of potato specific miRNAs was verified with miRPlant version 5 using default parameters.
Identification of phasiRNAs and PHAS loci was done with ta-siRNA prediction tool developed by Chen, Li, and Wu (2007) and both, the genome (PGSC_DM_v4.3) and the transcriptome sequences of potato with annotations from Ramšak et al. (2014). Minimum and maximum lengths of sRNAs were set at 18 and 25 nt, and the p-value threshold at 0.0001.
To identify vsiRNAs, first, the quality filtered reads from one of PVYNTN-infected samples were mapped to the sequenced genome of PVYNTN (isolate NIB-NTN, accession no. AJ585342) to obtain the consensus PVYNTN genome sequence infecting the plants. This was done to account for possible emerged mutations between our and the sequenced isolate of PVYNTN as described by Kutnjak et al. (2015). Second, the quality filtered reads of lengths 20-24 nt from all PVYNTN-infected samples were mapped to the reconstructed consensus PVYNTN genome using CLC Genomics Workbench (http://www.clcbio.com/) allowing only 100 % identity.
Genome_build: Solanum tuberosum DM genome (PGSC_DM_v4.3) available at http://solanaceae.plantbiology.msu.edu/pgsc_download.shtml
Genome_build: StNIB potato transcriptome available at http://www.gomapman.org/export/input/stNIB-v1_data
Genome_build: PVYNTN (isolate NIB-NTN, accession no. AJ585342) available at http://www.ncbi.nlm.nih.gov/nuccore/54021378
Supplementary_files_format_and_content: Tab-delimited text files include raw counts and RPM values of all identified small RNAs.
Supplementary_files_format_and_content: Consensus PVYNTN genome sequence in FASTA format.

Submission date

Jul 26, 2016

Last update date

May 15, 2019

Contact name

Marko Petek

E-mail(s)

[email protected]

Organization name

National Institute of Biology

Department

Department of Biotechnology and Systems Biology