Presentation 1: Nanopore Sequencing

Question 1

What are the advantages of Nanopore? (6)

Answer 1

1) Long Reads
2) Portable
3) Low up front investment
4) High throughput
5) Multiplexed library prep
6) Run multiple flow cells at the same time

Question 2

What are the disadvantages of Nanopore? (5)

Answer 2

1) Low per base accuracy
2) Requires high quality input data
3) Resource intensive data analysis
4) Fast changing, new technology
5) Introduce Indels

Question 3

What can Nanopore do that microarrays cannot?

Answer 3

Subtype gliomas into IDH mutant vs IDH wiltype samples and distinguish cancer entities from different tissue origins in real time within a few hours
Can detect novel mutations

Question 4

What are microarrays used for?

Answer 4

Targeted NGS panels that detect mutations in brain tumours that take several days
Microarray methylation based profiling takes up to 2 weeks
Only known mutations tested for

Question 5

What is low pass whole genome sequencing?

Answer 5

Untargeted sequencing
Requires the use of a reference genome for alignment
Lower cost than full coverage WGS
Provides a general picture of the entire genome
Low resolution
Cannot use for SNV analysis
Read distribution is random and dependent upon the reference genome’s compatibility
Often times less than 1x coverage

Question 6

Why would you use low pass whole genome sequencing?

Answer 6

This is enough information to get an overall picture of a genome​
Can look for global methylation, deletions or additions, and copy number in chromosomes

Question 7

How was Nanopore used to detect Copy Number Variation and Structural Variation?

Answer 7

Reads were aligned to reference genome​ and reference genome subtracted from LP-WGS alignments​. Large deletions or additions to chromosomes visible​. Long reads may span multiple copies, or junctions where insertions/deletions have occurred

Question 8

What was observed from Copy Number Variation and Structural variation?

Answer 8

WGS data used to generate CN profiles​Long reads cause some alignment artifacts . CN profiles closely resembled the matched SNP array-based profiles. Amplifications were seen, but deletions were missed.
Co-deletion of chromosome 1p/19q was detected

Question 9

How was methylation detected by Nanopore?

Answer 9

Because the native DNA molecule is being read by the pore, methylation information is retained in the squiggle
Methylated C causes a short pause in the reading of the strand, evident in the squiggle
These methylation events matched the methylation events in the methylome microarray so IDH mutant samples were correctly identified from Nanopore

Question 10

How was amplicon sequencing performed by Nanopore?

Answer 10

PCR used to amplify regions of interest for diagnosis. Extremely high sequence depth (>1000x)​. Very accurate consensus generation​. Real time monitoring of sequence depth. Amplicons also analyzed using Caliper LabChip for comparison

Question 11

How were SNVs determined using Nanopore?

Answer 11

Requires accurate full coverage reads Q20+ (i.e. cannot use LP-WGS)​. SNP profiles from each amplicon used to generate profile for patients. Extremely high sequence depth allows for greater accuracy​, so used deep amplicon sequencing. Nanopore amplicon data compared to microarray profile for validation of the workflow . The amplicon panel covered hotspot exon in IDH1, IDH2, HF3A, TP53, and pTERT. Real time analysis for monitoring read depth and stop sequencing when enough data present . Overall coverage >1000X within 2 to 20 minutes when using single run real time sequencing​