Lecture 2 : NGS libraries and basic data quality control
Modern Genomic Technologies  (LF:DSMGT01 )
Vojta Bystry
vojtech.bystry@ceitec.muni.cz

De-multiplexing
2
•Bcl2fastq tool
‒Needs sample sheet with indexes
‒Number of barcode mismatches
•Check undetermined

NGS data analysis
3
3
Raw data
.fastq
Genome/Transcriptome Reference Mapping
.bam
Interaction
analysis
CHIP-seq
Expression analysis
RNAseq
Variant analysis
WES
de-multiplexing
>
Not known
reference
QC
QC
Experiment design
Not ”classic” reference
Metagenomics
Reference assembly
Immunogenetic
VDJ-genes
CRISPR
sgRNA
Methylation
Bisulfide-seq
…

Primary data – fastq file
4


Fastq format - quality
5
•Fastq - q stands for quality – coded phred score
•
•
Quality
Error probability
5
31%
10
10%
20
1%
30
0.1%
•Very good for early problem detection
•Reasonable for trimming and read filtering
•RNA seq  - above phred score 5
•
CFFFFEFFGCEEGECFGGGGAFF87@E:++6C<++3:,8,33,,:,,,:,,:,,,

Fastq – quality control
6
•Fastqc - tool

•
•
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FastQC Report
7
Timeline Description automatically generated Table Description automatically generated

NGS data analysis
8
8
Raw data
.fastq
Genome/Transcriptome Reference Mapping
.bam
Interaction
analysis
CHIP-seq
Expression analysis
RNAseq
Variant analysis
WES
de-multiplexing
>
Not known
reference
QC
QC
Experiment design
Not ”classic” reference
Metagenomics
Reference assembly
Immunogenetic
VDJ-genes
CRISPR
sgRNA
Methylation
Bisulfide-seq
…

9
www.ceitec.eu
CEITEC
@CEITEC_Brno
Vojta Bystry
vojtech.bystry@ceitec.muni.cz
Thank you for your attention!

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