BIO00076H: Sequence Analysis - PowerPoint Presentation

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BIO00076H: Sequence Analysis - PPT Presentation

Lecture 2 Highthroughput sequencing Part 1 Kanchon Dasmahapatra kanchondasmahapatrayorkacuk Room J101 Workshop 1 trees mtDNA NJ tree TPI NJ tree a Are there differences between the mtDNA ID: 933344

151m chr10 hj772bbxx chr31 chr10 151m chr31 hj772bbxx 1101 sequencing 75s76m 1332 72m79s j00125 150m fastq genome 140 illumina

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Slide1

BIO00076H: Sequence AnalysisLecture 2High-throughput sequencing Part 1

Kanchon Dasmahapatrakanchon.dasmahapatra@york.ac.ukRoom J101

Slide2

Workshop 1 trees

mtDNA

NJ treeTPI NJ treea) Are there differences between the mtDNA and nuclear trees? b) Why might there be differences between these trees c) Is there any evidence for cryptic species? If so, how many cryptic species do you think there are? d) What evidence would you need to determine whether or not these were truly different species?

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L2 + W2: Learning objectivesBe aware of the current main Next Generation Sequencing technologiesBe aware of the advantages and disadvantages of each technologies Understand the FastQ

sequence output from these technologiesKnow how to quality check and clean FastQ filesHave a understanding of de novo assemblyHave a understanding of resequencing genomes

Slide4

A brief history of sequencing: 1977-1988

Sanger sequencing(radioactive chain-termination)

ACGTACGTGTTATCAGTACAT

Slide5

A brief history of sequencing: 1988-2002

Sanger sequencing(fluorescent chain-termination)

3 hours for 100kb

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Slide7

A brief history of sequencing: 2002-2019

NovaSeq

ABI SOLiD

Slide8

Illumina sequencing

https://www.youtube.com/watch?v=fCd6B5HRaZ8

Slide9

Illumina sequencing

Advantages

Very cheap per bpHuge output: 600Gb/dayLow error rateDisadvantagesShort read lengths: 2 x 150bpSequencer machine is very expensiveMain uses: Re-sequencing genomes of populations (people, mice, bacteria, viruses, yeast, fish, fruit flies, Plasmodium, Leishmania*)de novo assembly of small genomes (bacteria etc)polishing (correcting) long-read assemblies (PACBIO, ONT)Many other uses in functional genomics: RNAseq, ChipSeq, 3C, BarSeq, TnSeqMetagenomicsIllumina sequencing is currently the workhorse of most genomic and functional genomic projects.* You will get your hands on some Leishmania data

Slide10

IonTorrent

works by detecting the voltage (pH) change in wells as bases are sequentially washed over immobilised fragments, and then incorporated into a complementary DNA strand. It is similar to Illumina, and old-school Sanger sequencing in that it uses ‘sequencing by synthesis’

Slide11

IonTorrent

Advantages

Sequencer relatively cheapRelatively fast (4-7 hrs)DisadvantagesHomopolymer errors400bp sequence length1.2 – 2 GbMain uses: Small sequencing projects. Microbial genome sequencing. MetagenomicsHow IonTorrent works:https://www.youtube.com/watch?v=WYBzbxIfuKsIon Torrent is seldom used now(I have never used it).But the market changes all the time.

Slide12

PacBio sequencing

See an advert from Pacific Biosciences:

https://www.youtube.com/watch?v=v8p4ph2MAvI PacBio sequencing works by incorporating modified, fluorescently labelled, DNA bases in un-fragmented DNA, and detecting the flash of light.

Slide13

PacBio sequencing

Advantages

Long read lengths 10-15 kb; 40 kbDisadvantagesHigh error rates: 1.7%Sequencer is expensiveModest sequence output Main uses: Genome assembly, transcriptomes

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Nanopore sequencing

Oxford

Nanopore TechnologySee: https://nanoporetech.com/how-it-works

Slide15

Nanopore sequencing

Advantages

Very long read lengths 3 kb; 28 kb (2015) 7.5 kb; 117 kb (2016)Portable (has been used in caves)Sequencer is cheapIn rapid development nowDisadvantagesHigh error rates: 2-13%Expensive per bp of sequence (but coming down, close to Illumina)Main uses: Microbial genomicsGenome assembliesTranscriptomesOxford Nanopore Technology (ONT) is in very rapid development right now. Sequencing is being transformed (again!).

Slide16

Nanopore sequencing. What next?

The PromethION machine48 flow cells3000 nanopores

per flow cellHuge output promised, almost delivering.At the moment Nanopore technology is developing very fast.Output is increasing, error rates decreasing.Weirather et al. (2017) Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis. F1000Research, 6:100.

Slide17

Sequencing nowAt this point in time this is what we do:

Oxford Nanopore One of the technologies of choice for genome assemblies

.Produces the longest readsHas the worst error rateIs developing very fastIllumina:Technology of choice for genome re-sequencing (of populations)Widely used for small de novo genome assemblies, RNA-seq (sequencing transcriptomes), chip-seq, 3C, metagenomicsPacific BioSciences (PACBIO)One technology of choice for genome assemblies.Produces fairly long, fairly accurate readsSanger sequencing Using ABI machines.Technology of choice for low- to medium output sequencingEg: checking plasmids, small-scale population surveysNext year this slide will be different!

Slide18

Read more.Read more about the ultra-competitive world of NGS here: https://labiotech.eu/medical/ngs-dna-sequencing-illumina-qiagen/And about long read sequencing here:

https://doi.org/10.1016/j.tig.2018.05.008

(Also in the Paperpile collection, see the VLE)

Slide19

Fastq files and FastQCNext/3rd gen sequence produce ‘

fastq’ filesBases and a measure of the quality of each baseQuality scores enable users to check the quality their reads, and get rid of bad quality data

Slide20

Fastq file ‘anatomy’

Information about the fastq format and Phred-scores at:http://en.wikipedia.org/wiki/FASTQ_format

http://en.wikipedia.org/wiki/Phred_quality_score

Slide21

40 billion bases….what do I do with them?Fastq files(reads)

Check average quality statistics FastQC

Clean/trim sequencescutadapt, Reaper, Trimmotomaticde novo assembly SGAVelvet, CLCbio, and many othersCompare to other sequences/genomesBLAST, Cactus Genome-alignmentAnnotate (locate and mark up the genes)AugustusRemovePCRDuplicatesPicardtoolsSamtoolsAlign to areference genome ortranscriptomeBWANextGenMapNovoalignSNP and indel ‘calling’GATK, Samtools, Freebayes

Make vcf files (variant call format)Structural variant ‘calling’Delly, GenomeSTRiP, LumpyBiological analysisvcftools, AdmixtureMany, many othersVCF filteringbcftools, vcftools, GATK

Slide22

FastQC: quality filtering

GOOD SEQUENCES BAD SEQUENCES

Slide23

FastQC: quality filtering

GOOD SEQUENCES BAD SEQUENCES

Slide24

FastQC: quality filteringGOOD SEQUENCES BAD SEQUENCES

http://www.bioinformatics.babraham.ac.uk/projects/fastqc/Help/3%20Analysis%20Modules/

Slide25

Cleaning the dataCutadaptTrimmotomaticThese programs allow you to trim away poor quality sequence, and remove adapter contamination

Slide26

40 billion bases….what do I do with them?Fastq files(reads)

Check average quality statistics FastQC

Make vcf files (variant call format)Structural variant ‘calling’Delly, GenomeSTRiP, LumpyBiological analysisvcftools, AdmixtureMany, many othersVCF filteringbcftools, vcftools, GATK

Slide27

de novo assemblyEkblom & Wolf (2014) A field guide to whole-genome sequencing, assembly and annotation. Evolutionary Applications

7: 1026-1042.

Slide28

Slide29

Whole genome ‘resequencing’Usually used to collect information about diversity within a species (other uses also possible)Mapping/aligning to an existing reference genomeComputationally easier than de novo

assemblyMuch cheaper than de novo assemblyThese projects can have impressive scales:The 100,000 Genomes Project

has sequenced 100,000 genomes from around 70,000 people. Participants are NHS patients with a rare disease, plus their families, and patients with cancer.See: https://www.genomicsengland.co.uk/the-100000-genomes-project/ In workshops and group projects we will use some population sequencing data from the parasite Leishmania infantum.This data is unpublished – please do not share it.Read about this species here: https://en.wikipedia.org/wiki/Leishmania_infantum

Slide30

Whole-genome resequencing

Slide31

Whole-genome resequencing

Slide32

Aligning to a reference genomeDifferent aligners availableBWA: for less divergent sequences (fast)

NextGenMap: for more divergent sequences (slow)SAM: Sequence Alignment/Map format(https://samtools.github.io/hts-specs/SAMv1.pdf)

BAM is a compressed SAM file

Slide33