Similar protocols

Pipeline publication

[…] four types of samples from plants grown in greenhouses to the S2 stage: fertile anthers of LM15, sterile anthers of LM15RMs2, pistils of LM15 and LM15RMs2, and flag leaves of LM15 and LM15RMs2. Tissues were stored in liquid nitrogen during collection and then at −80 °C for storage. There were three biological replicates per tissue-genotype., We extracted total RNA using TRIzol (Invitrogen) and evaluated the RNA integrity with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The Berry Genomics Company prepared the sequencing libraries (ca. 500 bp per insert) and performed high-throughput sequencing (125 bp PE reads) using HiSeq2500. We pre-processed raw reads using the FastQC algorithm (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and then performed quality trimming and adaptor removal by Trimmomatic. In total, we retrieved 484.25 million clean pairs (), and used them for mapping and transcript assembly. For the PG5 gene, specific reads were indicated by a perfect match to PG5P1593S or PG5P1076F of LM15RMs2 using the STAR programme and a customized Perl script., On the basis of the current wheat genome, clean PE reads were mapped to a repeat-masked data set using the STAR programme. The PE reads were divided into three groups: (1) unique mapped reads—those mapped to a unique location in the masked genome, (2) multiple mapped reads—those mapped to at least two locations in the masked genome, (3) unmapped reads—those absent in the masked genome or those mapped to repeats (). Unique mapped reads were retrieved from the STAR-derived BAM files using Perl scripts., Based on the wheat genome (IWGSC1.0+popseq.28, http://plants.ensembl.org), we assembled the unique mapped reads using htseq-count script (v.0.6.1) with the union mode in the HTSeq package, and quantified their normalized transcript counts using the DESeq package (v.1.26.0). There were 79,153 genes with at least one nonzero count value in the four types of samples. To remove false positives and poorly-expressed genes, we defined a count cutoff value of 1.06 (). The count values less than 1.06 were assigned a zero value; 72,499 expressed genes were retained (). Between male-fertile (MF) and male-sterile (MS) anthers, we determined 7,294 differentially expressed genes (DEGs) by controlling the false discovery rate (FDR<0.01) for multiple testing in DESeq (v.1.26.0) and using the ajdusted P value (Padj<0.01). Furthermore, we divided the anther-related DEGs into two groups: (1) sterile-anther-enriched genes (SAEG): those associated with higher transcription in sterile anthers compared to the fertile anthers; (2) fertile-anther-enriched genes (FAEG): those associated with higher transcription in fertile anthers compared to the sterile anthers. The following rules were used to define the final list in each group: (1) genes active in only one type of anther with a count value >the 10th percentile (3.29) of the cleaned data (); (2) genes active in both MF and MS anthers but the relative amount was >10 fold in either MF or MS anthers […]

Pipeline specifications

Software tools FastQC, Trimmomatic, STAR, HTSeq, DESeq
Organisms Triticum aestivum, Hordeum vulgare
Diseases Infertility