How to know if mapping to reference genome was succesful?

Question: How to know if mapping to reference genome was succesful?

10 months ago by

eurioste • 40 wrote:

Given a set of BAM files for single-end reads just mapped with BWA tool, how can I have an overview if the mapping will result in useful information and the quality of the mapping was good? These data is to be used in a ChIP-seq experiment. Because this is a pilot experiment there are no technical duplicates, only one BAM for the input and for each ChIPed proteins/histone mark. Because of this a "Correlation among samples" plot will not be useful.

I'm new to NGS and ChIP-seq data and I tried to find useful tutorials/ references for galaxy but could not find one dealing with a dataset in a realistic manner.

Which tools and plots should I use? Any general workflow for assessing mapping quality would be appreciated.

EDIT: I would like to have something like the distribution of MAPQ values or the distribution of the reads along the chromosomes.

mapping bam chip-seq • 366 views

ADD COMMENT • link •

modified 10 months ago • written 10 months ago by eurioste • 40

10 months ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

QA/QC is covered in the Galaxy tutorials here: https://galaxyproject.org/learn/

Try these to start with (for assessing sequencing and mapping quality):

NGS logistics - this is an introduction to Galaxy's functionality for the analysis of Next Generation Sequencing data.
ChIP-seq: A simple ChIP-seq experiment with two replicates - an example analysis for finding transcription factor binding sites.

Thanks! Jen, Galaxy team

ADD COMMENT • link written 10 months ago by Jennifer Hillman Jackson ♦ 25k

Thanks for the answer but unfortunately these tutorials don't teach what I need. I've got got FastQC and pre-processing well covered and the tutorials only deal with deduplication and cross-sample correlations in post processing. I would like to have something like the distribution of MAPQ values or the distribuiton of the reads along the chromosomes.

ADD REPLY • link written 10 months ago by eurioste • 40

Hello,

This section related to bigWig coverage should help: https://galaxyproject.org/tutorials/chip/#generating-bigwig-datasets-for-display

And this tutorial has more help for assessing mapping success: http://galaxyproject.github.io/training-material/topics/chip-seq/tutorials/tal1-binding-site-identification/tutorial.html#step-7-inspection-of-peaks-and-aligned-data

Downstream tools can also be used to generate stats/metrics. Search for bigwig in the tool panel to find these - many are in the DeepTools tool group. Example: computeMatrix followed by plotHeatmap and/or plotProfile. More usage details are on the tool forms.

MAPQ stats can be pulled out into a tabular dataset and graphed. Convert BAM-to-SAM as needed (no header). Use Cut to extract the MAPQ value (5th column). This is now a tabular dataset that can be used with Charts (found under the Visualize icon, per dataset (the small graph icon) ). Charts can be used with any tabular data input - so any numerical value you are curious about could be isolated in a similar way and graphed.

Hope that helps with more options!

ADD REPLY • link modified 10 months ago • written 10 months ago by Jennifer Hillman Jackson ♦ 25k

Similar posts • Search »