Next Generation Sequencing Part Two Flashcards
Overview sample library preparation.
- For any NGS platform we first need to make a library - what constitutes the library depends of what you want to do. If you want to sequence the whole genome of an individual then your starting point would be genomic DNA.
- Start with your input DNA which may be genomic DNA or enriched target areas.
- Fragment the DNA to a smaller size using nebulisation, shearing, or enzymatic digestion. Fragments are generated in the 150-600bp size range depending on platform and application requirements.
- Fragments have terminal overhangs which require blunt end repair and phosphorylation.
- Ligation of adapters.
- Purification of repaired fragments by spin column or magnetic beads.
- Hybridisation to sequences complementary to adapter sequences (emulsion PCR or bridge amplification).
- Amplification.
Outline the Truseq Custom Amplicon method of targetting genes of interest in NGS.
Illumina Truseq Custom Amplicon:
The TruSeq custom amplicon assay is a simple and streamlined method for capturing and amplifying targeted regions of interest.
1) . Use DesignStudio to create custom oligo capture probes flanking each region of interest.
2) . Probes hybridise to flanking regions of interest in un-fragmented gDNA.
3) . Extension/ligation between custom probes across region of interest then takes place.
4) . PCR adds indices and sequence primers.
5) . Uniquely tagged amplicon library ready for cluster generation and sequencing.
What methods can be used for target enrichment in order to sequence only regions/genes of interest in NGS library prep.
1) . Illumina Truseq Custom Amplicon
2) . HaloPlex
3) . Illumina Nextera Rapid Capture Custom Enrichment (large ROIs)
4) . Agilent SureSelect (large ROIs)
5) . PCR amplification by fluidigm acess array system (small ROIs).
6) . RainDance Microdroplet PCR
7) . Roche Numblegen - Solid-phase capture with custom designed oligonucleotide microarray.
8) . Long range PCR )fragment or use nextera).
9) . Multiplex PCR.
Outline the HaloPlex method of targeting genes of interest in NGS library prep.
HaloPlex:
1) . Digest and denature sample DNA - Sample DNA is first digested and denatured. Restriction enzymes are used to fragment DNA.
2) . Hybridise Oligonucleotide probe library - Each probe is designed to hybridise to both ends of a targeted DNA fragment, guiding the targeted fragments to form circular DNA molecules. The probes also contain a method-specific sequencing motif that is incorporated into the circularisation.
3) . Purify and ligate targets - HaloPlex probes are biotinylated so targeted fragments can be retrieved using magnetic streptavidin beads. Target-probe complexes are closed to ensure that only perfectly hybridised fragments are circularised.
4) . Amplify targeted fragments with PCR. Only circular DNA targets are amplified, providing an enriched amplification product that is ready for sequencing. Sample barcodes are introduced during amplification for precise tracking.
The HaloPlex PCR protocol combines target enrichment and library preparation in a single workflow. The current protocol takes two days to complete, however, alterations in the HaploPlex protocol can reduce the turnaround time to less than one day.
Outline the Illumina Nextera Rapid Capture Custom Enrichment method of targeting genes of interest in NGS library prep.
With Nextera technology, DNA is simultaneously fragmented and tagged with sequencing adapters in a single step, using standard lab equipment. Ideal for precious samples available in limited quantity, the protocol requires only 50 ng of DNA input.
- Nextera primarily for large regions of interest.
- Uses transposons which can simultaneously fragment and tag the DNA without the need to shear it.
- Up to 15Mb of the DNA can be targeted therefore this method allows for simulations targeting of many genes but is not suitable for less than 20-25 genes.
- Similar to Illumina Trusight method used in illumina off the shelf panels. Therefore has advantage of being able to run with Trusight samples on MiSeq.
Outline the Agilent SureSelect target enrichment method of targeting genes of interest in NGS library prep.
- Agilent SureSelect is another target enrichment method.
- Again this is for use with large regions of interest and requires the initial fragmentation of DNA and thus requires investment in a sonicator which can be expensive.
Outline the fluidigm access array target enrichment method of targeting genes of interest in NGS library prep.
- The fluidigm acess array system allows amplification of 48 primer pairs and 48 patients.
- The system allows for the incorporation of patient tags and instrument specific adaptors such as 454 or Iontorrent without a ligation step.
- The finished product is ready for amplification by emulsion PCR.
- An advantage of this method is that although PCR based the reactions take place in extremely small volumes thus saving on consumables.
Outline the RainDance Microdroplet PCR target enrichment method of targeting genes of interest in NGS library prep.
- Microdroplet PCR-based target enrichment.
- The process of merging pico titre volume droplets on fragmenting genomic DNA with primer pair droplets in a one to one ratio on a microfluidic chip to form PCR droplets.
- The resulting PCR droplet library consisting of over 1.5 million droplets is amplified to enrich for specific regions of the genome.
Outline the PCR target enrichment method of targeting genes of interest in NGS library prep.
- Long range PCR or multiplex PCR can be used for target enrichment.
- Have the advantage that no special equipment is required but can be labour intensive.
- Long range PCR has already been used to screen for BRCA1 and BRCA2.
- Instead of fragmentation Nextera technology can also be used to fragment and add barcodes.
What does the term read length mean?
Read length - the number of bases sequenced in a fragment.
What does the term capture efficiency mean?
Capture efficiency - what portion of the region of interest captured is on target and what portion is off target.
What does the term paired end sequencing mean?
paired end sequencing - sequencing from both sides/ends of the region of interest.
What does the term read depth mean?
Read depth = how many times has a based been sequenced.
Outline validation required for NGS.
- Coverage - is all of your region of interest sequenced. Do you need to do Sanger sequencing for the missing region?
- Required read depth?
- Can you detect indels, substitution mutations within any homopolymer tracks?
- Repeatability.
- Reproducibility.
- Documentation of analysis pipeline.
- BPGs.
What are the data processing considerations associated with NGS?
Data processing:
- Data storage.
- Sequence quality assessment.
- Alignment with ref seq/de novo.
- Algorithm to detect mutations; software cost, consider polys, UVS.
- Need for bioinformaticians.
