I'm looking to count RT stops for a sequencing job I performed. I have a reference sequence that I am mapping to. I am using the iterative mapping ===> count rt stops workflow from galaxy. My RT stops come out differently than when I use an alignment program and manually count. Also my RT stop counts differ greatly if my input is DNA or RNA of the same reference. Any help is appreciated.
Check the mapped BAM inputs. Does the workflow use the exact same input, tool, and parameter settings as when you executed mapping independently?
If not exact, then some differences would be expected. How different is related to all the input factors - some will have more impact than others.
Try to exactly reproduce in your history what the workflow is doing and the results should be the same. Note: perhaps not 100& identical, but those changes should be very small, just to do small rare (assumed to be insignificant) differences that can occur between mapping runs. (Whether executed in Galaxy or not)
Comparing data across different work/analysis paths is a great way to learn about tools and fine tune parameters. Maybe also consider setting up and executing a matrix of tests. One variable change per run, organized so that only one (or 2+ related) factors distinguish each test. The goal would be to maximize the common results and review differences to see which method is the best overall chose for your experiment.
The above is a very common practice (probably requisite, unless the original default workflow is acceptable for your use). It is so common and of such high interest that some publications are focused entirely or in part on offering insight and practical usage conclusions these sorts of comparisons produce. Nearly all bioinformatics people do this routinely as a standard step when building up a good experiment, whether the analysis is headed for publication or not, and whether that background research is intended to be included in the publication or not.
Galaxy is a great place to do this - all test executed would have complete reproducibility (inputs, tools, params, and other provenance information is automatically captured). Results can be easily graphed or further manipulated as part of data reduction steps. Plus any Galaxy object (History, Dataset, Workflow, Visualization, Page) can be shared with anyone (get a PDF/screenshot of workflow/history contents, or publishe a share link to objects, and the like).
Take care, Jen, Galaxy team
I am trying to get raw read counts of RT stops to a reference from a sequencing job. I'm using iterative mapping ===> count RT stops.
It seems to work and provides me with numbers for every position which seem to make sense. All I'm trying to do is find another way to confirm those stop counts. Most workflows are made for structural analysis (such as SHAPE reactivity) rather than raw read outs of RT stops. If there is another way to get raw counts of stops or that will map it for me and I can manually count the raw stops. When I use iterative mapping then count RT stops I'll get several hundred reads for some positions. Any other mapping program I use will have very few (lets say 1,000 from iterative mapping to 5 if I do it on bowtie then count). I'm trying to figure out why this is or what I'm doing wrong?
In other words would settings and programs would best confirm raw RT read counts that I get from iterative mapping ===> RT counts.