NGS methods and applications Flashcards
Purpose of library prep
3 main steps
Prepares DNA for sequencing, depending on NGS platform used
Fragmentation
Adaptor/indices ligation
Enrichment
Purpose of an enrichment step
Capture the ROI for single genes, targeted panels or clinical/whole exome
2 types of enrichment
Amplicon/PCR based
Hybridisation/Capture based
2 advantages of amplicon enrichment
Lower cost and faster TAT
Decreased amount of starting DNA
Name an example kit that uses amplicon enrichment and an application
QIAseq for somatic cancer panels
3 disadvantages of amplicon enrichment
Preferential amplification = non-uniform enrichment
Artefacts
Difficult to multiplex for high ROI numbers
What are UMIs?
Unique Molecular Indices are used to tag an original DNA frag. Allow filtering of PCR duplicate reads based on UMI. Can also be used to identify CNVs
Basis of capture enrichment
RNA or DNA oligonucleotides specific to target ROI and ‘pulled down’ using biotin and streptavidin labelled bead clean up
Creates a tile of captured fragments that represent a whole ROI
3 advantages of capture based enrichment
More uniform coverage and improved for GC rich regions
PCR duplicates are easily recognised and removed
Higher read depth can be achieved for CNV/mosaicism analysis
2 limitations of capture enrichment
More starting material required
High costs and slower TAT
1 example kit using capture enrichment
Agilent SureSelect
Describe short read NGS
Series of automatically coordinated repeated chemical reactions, carried out in a flow cell which has immobilised templates and reagents.
What is the main sequencing method for SRS and outline the process
Sequencing by synthesis
Repeated cyclical process involving nucleotide addition, washing and signal detection
Briefly describe Illumina SBS sequencing
Solid phase bridge amplification to clonally amplify templates to generate clusters, followed by single based sequencing using fluorescence
Briefly describe IonTorrent sequencing
Measures release of hydrogen ions when a nucleotide is incorporated into DNA by polymerase. The potential difference due to the pH change is converted to a base call by an ion sensor
Name 2 long read sequencing methods
Single Molecule Real Time (SMRT) sequencing
Nanopore sequencing
Name an application for SMRT
Rapid identification of infectious pathogens
3 steps of nanopore sequencing
- dsDNA is unzipped to form ssDNA that moves through the pore
- Flow of ions produces a current depending on the base in ssDNA passing through the pore
- Adaptor molecular keeps bases in place long enough to identify
2 advantages of SRS
High accuracy and throughput
Cost effective
4 advantages of LRS
Allows variant phasing by haplotype generation
Better SV and CNV calling
Can distinguish genes and pseudogenes
Sizing of repeat expansions
4 limitations of LRS meaning it is not applicable for diagnostics (yet)
Not cost effective
High error rate
Requires high molecular weight DNA
Not standardised
4 main steps for NGS bioinformatics
QC
Alignment
Variant calling
Annotation
3 files for NGS data storage
FASTQ (text file with base calls and QC data)
BAM (aligned data with variants/coverage info)
VCF (annotated variants)
2 sources of sequencing errors in NGS
Polymerase misincorporation
Cross talk from nearby cluster
