NGS analysis

What do you sequence?
1 Quiz
Applications
Library preparation
5 Topics | 1 Quiz
Steps in the library prep protocol
The basic library prep protocol
Protocol for targeted sequencing
ChIPSeq libraries
RNASeq libraries
RNASeq libraries
Illumina sequencing
8 Topics | 2 Quizzes
Flow cells
Forming clusters on the flow cell
The sequencing reaction
Sequencing steps
Detection of the sequencing signal
Illumina sequencing generates short reads
Paired end sequencing
Paired end sequencing
Illumina sequencers
Benefits of paired end sequencing
Hardware requirements for NGS analysis
Illumina data formats
5 Topics | 2 Quizzes
raw Illumina data
Illumina data
FASTQ files
Quality scores
FASTQ files for paired end experiments
FASTQ files are sometimes messy
The data sets for this course
NGS workflows
Platforms for NGS analysis
2 Topics
Galaxy
Galaxy interface
Coverage
Quality control of FASTQ files
10 Topics | 5 Quizzes
Quality control of FASTQ files
FastQC to check the quality of the reads
The basic statistics module
Basic statistics
Per base sequence quality
Per base sequence quality
Per sequence quality
Per tile quality
Per tile quality
Duplicates: why are they an issue?
Sequence duplication levels
Duplicate sequences
Overrepresented sequences
Overrepresented sequences
Base and GC content
Improving the quality of the data
3 Topics | 1 Quiz
Improve the quality of the data
Filtering and trimming
Trimming low quality bases
Trimming low quality bases
Mapping DNA reads
7 Topics | 3 Quizzes
Goal of the mapping
Variants versus sequencing errors
Workflow for mapping DNASeq reads
Download the reference
Format of reference?
Index the reference
Mapping single end versus mapping paired end reads
Tools for mapping
Output of the mapping: bam/sam files
Cigar strings
Mapping RNA reads
9 Topics
Goal of mapping RNASeq reads
Workflow for mapping RNASeq reads
Download the reference files
Built in references in usegalaxy.be
Mapping to the genome or to the transcriptome
Overview of RNASeq mappers
Mapping to the genome
Mapping to the transcriptome
RNASeq workflow for SE and for PE data
Processing the mapping results
5 Topics | 1 Quiz
Processing the mapping results
Marking duplicates
Processing bam files for use in GATK (variant calling software)
vcf format
Interpretation of vcf files
Processing RNASeq mapping results
Checking the quality of the mapping
4 Topics | 1 Quiz
Checking the quality of the mapping
RSeQC Genebody coverage checks RNA degradation
RSeQC: other tools
Potential issues to check after mapping RNASeq reads
Issues in RNASeq data
Visualization of mapping results
1 Topic
Visualization of RNASeq mapping results
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Output of the mapping: bam/sam files

NGS analysis Mapping DNA reads Output of the mapping: bam/sam files

What are bam files?

Mappers will produce either a sam or a bam file. What’s the difference?

Content-wise there is no difference: they contain exactly the same info. But there is a difference in format:

  • sam files are tab delimited text files
  • bam files are binary files

Binary files are very compact, but you cannot open them in a text editor. Your computer can read them though.

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