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Slamdunk (version 0.4.3+galaxy1)
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Multimapper recoveries
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Advanced settings.s
Advanced settings. 0
Number of T>C conversions required to count a read as a T>C read (default: 1).
Maximum read length (before trimming).

SLAM-seq

SLAM-seq is a novel sequencing protocol that directly uncovers 4-thiouridine incorporation events in RNA by high-throughput sequencing. When combined with metabolic labeling protocols, SLAM-seq allows to study the intracellular RNA dynamics, from transcription, RNA processing to RNA stability.

Original publication: Herzog et al., Nature Methods, 2017; doi:10.1038/nmeth.4435

Slamdunk

To analyze a given SLAM-seq dataset with slamdunk without recovering multimappers, you only need to provide the following files and keep everything else to the default parameters.

Parameter Description
Genome The reference fasta file (Genome assembly).
Reference BED-file containing coordinates for 3' UTRs.
Reads Sample FASTQ(gz) files.
Read length Maximum length of reads (usually 50, 100, 150).

This will run the entire slamdunk analysis (slamdunk all) with the most relevant output files being:

  • Tab-separated tcount file (Count TSV) containing the SLAM-seq statistics per UTR
  • BAM-file with the final filtered mapped reads
  • VCF file of variants called on the final filtered alignments

These files can be input to the Alleyoop tool for visualization and further processing. See the Slamdunk documentation for more information.

Multimapper recovery

To utilize multimapper recovery, modify the following parameters. You must either choose a separate 3' UTR file or activate filtering on the supplied reference file. Will only yield different results than a unique-mapping run by specifying a number > 1 as maximum number of multimapper aligments to consider.

Parameter Description  
Maximum number of alignments to report per read The maximum number of multimapper alignments to consider.
Use separate 3' UTR bed to filter multimappers. 3' UTR bed file to filter.
Use reference bed file to resolve multimappers. Use reference as 3' UTR bed file to filter.

T>C conversions

Depending on the use case, more stringent or more lenient measures of T>C conversion and T>C reads are required such as 2 T>C by Muhar et al., Science, 2018; http://doi.org/10.1126/science.aao2793

This can be controlled by the following parameter:

Parameter Description  
T>C conversion threshold Minimum number of T>C conversions to consider a read as T>C read.