Long Read Sequencing Meeting

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1. Whats new in long read sequencing at NGI

national genomics infrastructure (uppsala and stockholm)
certified service provider of pacbio
10x read linked-read sequencing
Hi-C kits early testing
ONT - under evaluation currently - want to provide it also as a service
HMW DNA, data management and analysis
Long Read club is online club for this

1.1 SweGen

seq. Of 1000 genomes with different methods.
they use all the data and combine it
identify lots of unknown regions
over 10Mb not presented in human ref. Incl. Worm DNA
why long read in clinical use:
publication about long read seq. In clinic

2. Technologies for long reads

2.1 10x Genomics, overview

https://www.10xgenomics.com/

they have single cell and bulk genomic technologies
open source free software pipeline
gele bead emulsion, 8 channels for 8 samples
micro reaction vessels (gem)
gem: cell + enzyme + gele bead
Linked read libraries
basically it's like multiple barcoding and tagging steps to solve some issues of short assembly
they tag reads so it's clear from wich HMW DNA the read is originating from
e.g. 2 repeat regions, reads from region A are taged red. Region B reads are tagged in blue

2.2 Oxford Nanopore, latest

engineered nanopore, did lot of point mutations
basecaller to call variations (?), they have specific caller?
flongle: 1 Gb in 16 h
200Gb per promethion flowcell
24 cell run 3 Tb (125 Gb per cell)

User publications

Nanopore "private Q&A"

Flipflop with a methylation aware will come soon
use Tombo in github for now to get methylation info
refueling step will be soon implemented via software, tells you when to do the step
flongle runs only for around 16h with around 1 Gb
ubuntu18 guppy soon, no GPU minknow support for live basecalling
nuclease wash protocol on nanopore site
only useful for fast pore dying, not after a big run

2.3 PacBio, latest

Accuracy

89.0 % read accuracy (over 20kb)
circular consensus sequencing
increased to 99.8% with PacBio HiFi
- but less than 20kb reads
basically to improve read quality PacBio needs to decrease total read length. (Circular read consensus)
15x coverage seems to be enough, more is not increasing quality or accuracy

Updates

improved lib prep protocol for low input
100ng starting input (support at 150ng)
starts mid April
target enrichment using crispr/cas9
q3 2019 (2 day protocol)
RNA still based on RT-PCR

New sequel II system

8x yield
lower cost
accuracy seems to bit a smaller bit better than previously
they claim that it's now getting better than short reads

3. Topics and speeches

3.1 INRA ENSAT

Mohamed-Zouine, INRA/ENSAT. Long DNA molecule sequencing: from de novo assembly to chromosome level scaffolding of the tomato and melon genomes

tomato and melon genomes
tomato: 12 chromosomes, 950Mb genome
they Mix pacbio bionano and 10x genomics
bionano is optical mapping
chromium 10x was used
have protocol for high gDNA
annotation via EugenEP pipeline
TomExpress for tomatoes
jbrowse to explore annotations
reproduced the results of their pipeline with a melon
currently doing "phylogeny" on different tomato species
include now also ONT

3.2 Targeted sequencing

Ida-Höijer, UU. New approaches for long read targeted sequencing

"fishing the genome"
usually via amplification
CRISPR-Cas9 system for amplification free targeted sequencing from pacbio
seq lib. Prep. Like normal
cas9 digestion only on pac dna with gene of interest (target)
then close these open DNA fragments with gene of interes with another adapter
so all circular pac reads with the DNA of interest are now marked
why no amplification ?
different repeat regions length of same target would create strange results in PCR
these usually come from PCR errors.
so you wouldn't know what's the cause of this
but with crispr cas9 you know that the target has different lengths
Xdrops target enrichment
droplet of DNA fragments
pcr and labeling of each droplets
sorting of these via FACS

3.3 Human genome variant calling via pacbio

Alexander-Hoischen Radboud UMC. Medical genetics: Identification of hidden structural variants with long read sequencing

www.SOLVE-RD.eu solving rare diseases
2 people differ in over 4 mio positions
28Mb of human genome is only covered via long reads
pacbio Sv determination
10 of the SV are Not represented in the Shorts read data
100 inversions per Genome (validation tricky)
they can detect SNVs, they are waiting for the new pac bio

3.4 Long reads f. personalized therapeutics

Yahya-Anvar, Leiden University Medical Center. Long reads paving the way to personalized therapeutics

genetic factors vary the drug response
the human genome is complex and incomplete
2 gene's, fully duplicated in the genome are responsible for around 80 % of the current drug response
average pacbio length is 13kb in their experiments (big peak there) nothing really above or below

3.5 Human Genome Sequencing on nanopore

Wigard Kloosterman, UM Utrecht. Unraveling human genome structure using long read Nanopore sequencing

chromothripis, Chromosomen Break and reattachment (reshuffling basically)
question was if nanopore can detect this
direkt Sequencing of long pcr products
SV calling using long reads
nanoSV a pipeline to call SVs
14x coverage is needed for SV calling
showed a crspr-cas9 enrichment was shown. Similar to the pac bio approach: cut and special adapter add

3.6 Hybrid assembly honey bee fly

Matt Webster, Uppsala University. A chromosome level hybrid assembly of the honeybee genome

really good data and benchmarks
pacbio, 10x genomic linkage, bionano

APPROACH

wtdbg2 + racon + medaka + pilon (10x genomes)

3.7 Norway sprouch / pine trails

Niklas Mähler, Umeå University. Long read assembly of Norway spruce and Scots pine

they need a reference genome
they want to decrease the breeding times via genomic selection
10 mio scaffolds on the 2013 "WGS 1.0"
WGS 2.0:
50x pacbio genomic DNA
N50 12kb
removed 0.58 % chloroplast and 7.9 % mitochondria DNA

Software

wtdbg2 & marvel marvel * all vs all alignment, no error correction, masks repeats - 150000CPU for one algo round * they need 2 mio CPU hours and 1 Pb of storage for pines wtdbg2 * used reads longer than 12kbs on 15x coverage * needs lots of RAM * they did busco on wtdbg2, bad results * however the 10 mio scaffold aligned nicely * they have currently "hardware" issues with CPU time and storage due to the size

3.8 SMRT analysis on plasmid genes content and methylation profiles

Åsa Sjöling, KI. SMRT analysis of plasmid gene content and methylation profiles within clonal lineages of enterotoxigenic Escherichia coli (ETEC)

E. coli plasmids
interpulse duration for methylation. The lag in measurement of the base is detected
plasmids harboured the methyltransferase M.EcoGIX, usually in IncI and IncF
protects the plasmid of being degrated in another host

3.9 Nanopore seq. Gene Fusion and alternative splicing

Wilfried Haerty, Earlham Institute. Characterization of splicing diversity and gene fusions through Nanopore sequencing

human (19.836 Protein coding sequences)
lots of different splicing types
example here is calcium channel
expressed in various cell types
risk gene for multiple disorders
they want all isoformes now
mRNA over 13kb
they only have exon information. Not what's the isoformes?
analysed various brain regions, so different tissues
found 90 isoformes, 7 previously annotated, 83 new
the top 10 isoformes account for 75 % of transcripts
tissue variation is way greater than between individuals
51 of the 83 new are coding

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