Frequently asked questions

Why am I getting a warning about possible low-quality reactivity profiles?

The run has failed to meet one or more run quality checks. Check the log file to see which check(s) failed, and refer to Quality control checks for explanations and possible solutions.

How should I handle primer trimming?

Random primers

If a Nextera or similar prep is used, no explicit primer trimming should be necessary, since the ends of input cDNA molecules will largely be digested away before sequencing.

Otherwise, the length of random primers used should be provided to ShapeMapper using --random-primer-len.

Amplicon primers

For most users, simply set primer binding sites to lowercase sequence in the input .fa file, and run ShapeMapper with the --amplicon option. This will handle cases with a single pair of primers on either end of the target sequence.

For more complex cases with internal PCR primer locations or multiple pairs of primers, run ShapeMapper with a primers file input with --primers <primers_file> . ShapeMapper will automatically determine the expected locations of input PCR primers by comparing their sequences with those of reference targets.

Multiple amplicon primer pairs within a single dataset are also supported. Some groups use a tiled amplicon strategy to cover low-abundance target RNAs.

For additional details on primer trimming and read location requirements, see Primer trimming.

How do I model RNA secondary structures?

see Other software

How do I render SHAPE reactivity-colored secondary structures?

see Other software

Can I run ShapeMapper on multiple RNAs at the same time?

Yes. The --target parameter will accept multiple FASTA files, or multiple sequences can be included within a single FASTA file.

However, inputting large numbers of sequences (such as those from whole-transcriptome studies) will cause ShapeMapper to run out of memory or crash. The shapemapper-txome wrapper can be a useful workaround in this case, or see Modular workflow for help running alignment parsing, mutation counting, and reactivity profile calculation steps in isolation.

Can I run ShapeMapper on an existing alignment?

The standard shapemapper executable does not currently accept alignment files as input. However, individual shapemapper modules can be run "manually" to process alignments in SAM format (see Modular workflow for guidance).

What should I do if I am studying an RNA transcribed from multiple loci with slightly different sequences?

The presence of a subpopulation of sequence variants or transcribed pseudogenes is often initially indicated by an unusual number of nucleotides with high apparent mutation rates in the untreated control sample.

If the sequences of contaminating/alternate transcripts are available, we recommend providing all sequences to ShapeMapper (the --target FASTA file can have multiple RNAs within it, or multiple FASTA files can be provided). There is currently no good fully automated tool for determining the sequences of contaminating transcripts - this would require a sequence assembly, which remains an art, especially with noisy reads from reverse transcription.

Care should be taken that identical sequences are not provided to ShapeMapper, as this will result in reads aligning to multiple targets with similar alignment scores and therefore failing to meet --min-mapq.

If sequences are not available, the --render-mutations ShapeMapper option can be useful in some cases. This will render individual reads and their mutations (and debugging info that can be ignored) up to --max-pages. For short transcripts, this allows quickly spotting frequent patterns of mutations that result from the presence of contaminating transcripts. By default, the page size is scaled to include reads up to 800 nucleotides long. This is often too zoomed out for convenient visualization, so the --max-paired-fragment-length parameter may need to be lowered.

Warning: running ShapeMapper with more than 15 highly similar target sequences can exhaust bowtie2's search limits and result in mis-mapping across sequences. In this case, we recommend increasing the aligner's effort options with --max-search-depth and perhaps --max-reseed, corresponding to bowtie2's -D and -R options. See bowtie2 documentation.

Can I run ShapeMapper using reads from PacBio or other long-read sequencing platforms?

This is currently unsupported (see Read length).

How should I cite ShapeMapper?

For ShapeMapper2 software, please cite:

Busan S, Weeks KM. Accurate detection of chemical modifications in RNA by mutational profiling (MaP) with ShapeMapper 2. RNA. 2018, 24(2):143-148. link

For the MaP (mutational profiling) RNA adduct readout strategy, please cite either:

Siegfried NA, Busan S, Rice GM, Nelson JA, Weeks KM. RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP). Nat Methods. 2014, 11(9):959-65. link

Smola MJ, Rice GM, Busan S, Siegfried NA, Weeks KM. Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis. Nat Protoc. 2015, 10(11):1643-69. link

    

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