4/25/2023 0 Comments Rna sequence analysis![]() Although there is general agreement between the mappings and the gene quantifications produced by different RNA-seq pipelines, quantifications of individual transcript isoforms, being much more complex, can differ substantially depending on the processing pipeline employed and are of unknown accuracy. The mapping of the reads is done using the STAR program (in some cases, both STAR and TopHat aligners are used to produce separate bam files) and the quantification of genes and transcripts is done with the RSEM program. The pipeline also produces quality metrics, including Spearman correlation and read depth. Please see the caution regarding transcript quantifications in the paragraph below titled "Regarding alignment and quantification". column 9: posterior_standard_deviation_of_count.column 7: FPKM (fragments per kilobase of transcript per million).column 6: TPM (transcripts per million).The file format specifications are as follows: For unstranded data, signals are generated for unique reads and unique+multimapping reads without regard for strand identity. Produced by mapping reads to the transcriptome.įor stranded data, signals are generated for unique reads and unique+multimapping reads in both the plus and minus strands. Please see the paragraph titled "Regarding alignment and quantification" below the "Outputs" table for more on the aligners and their indices. The spike-ins are effectively the controls for the RNA-seq experiment. Please see the paragraph titled "Regarding alignment and quantification" below the "Outputs" table for more on the aligners and their indices.ĮRCC Spike-ins (External RNA Control Consortium) Reads must meet the criteria outlined in the Uniform Processing Pipeline Restrictions. View the current instances of this pipeline for single-ended data View the current instance of this pipeline for paired-ended data In the future, this pipeline may also be used to process PAS-seq and Bru-seq data. Libraries must be generated from mRNA (poly(A)+, rRNA-depleted total RNA, or poly(A)- populations that are size-selected to be longer than approximately 200 bp. The ENCODE Bulk RNA-seq pipeline can be used for both replicated and unreplicated, paired-ended or single-ended, and strand-specific or non-strand specific RNA-seq libraries. The full pipeline code is freely available on Github and can be run on DNAnexus (link requires account creation) at their current pricing. The Bulk RNA-seq pipeline was developed as a part of the ENCODE Uniform Processing Pipelines series.
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