DE NOVO WHOLE GENOME ASSEMBLY AND SEQUENCING OF THE SUPERB FAIRYWREN. (Malurus cyaneus) JOSHUA PEÑALBA LEO JOSEPH CRAIG MORITZ ANDREW COCKBURN

Transcription

1 DE NOVO WHOLE GENOME ASSEMBLY AND SEQUENCING OF THE SUPERB FAIRYWREN (Malurus cyaneus) JOSHUA PEÑALBA LEO JOSEPH CRAIG MORITZ ANDREW COCKBURN

2

3

4

5

6 Synthetic Long-read (Moleculo)

7 Short insert shot-gun Mate-pair Synthetic Long-read (Moleculo)

8 Short insert shot-gun Mate-pair Synthetic Long-read (Moleculo) CHiCago

9 Short insert shot-gun Mate-pair RSII Synthetic Long-read (Moleculo) CHiCago

10 Short insert shot-gun Mate-pair RSII Sequel Synthetic Long-read (Moleculo) CHiCago

11 Short insert shot-gun Mate-pair RSII Linkage map 26K SNPs Sequel Synthetic Long-read (Moleculo) CHiCago

12 Short insert shot-gun Mate-pair RSII Linkage map 26K SNPs Sequel Chromium Synthetic Long-read (Moleculo) CHiCago

13 Short insert shot-gun Mate-pair RSII Linkage map 26K SNPs Chromium Sequel Chromium Synthetic Long-read (Moleculo) CHiCago

14 Short insert shot-gun Mate-pair RSII Linkage map 26K SNPs Chromium Sequel Chromium Saphyr Synthetic Long-read (Moleculo) CHiCago HiC

15

16

17

18 Historic DNA

19 Historic DNA Adaptation

20 Historic DNA Adaptation Structural variation

21 SO, YOU WANT TO SEQUENCE A REFERENCE GENOME? TYPES OF PUZZLE PIECES ACATCTAGATC ACTAGTCGATC GAGCTATCGAT CGATCGATGAT CGATCGATTGA 1 1. Ecology & Evolution, Australian National University, Canberra 2. Centre for Biodiversity Analysis, Canberra 3. Australian National WIldlife Collection, @ josh.penalba@gmail.com (population with lowest diversity) Female (ZW) DNA quantity 2n = 72 ~1.1Gb Long distance placement of puzzle pieces determined. High quality short fragments. SEQUENCE DATA DNA insert size: > 1kb SHORT READS 1,3 Malurus cyaneus Flinders Island Cost ACATCTAGATC T Illumina shotgun Great quality, small standard puzzle pieces. Comes in a small range of sizes. JOSHUA V. PEÑALBA, LEO JOSEPH, CRAIG MORITZ, ANDREW COCKBURN 2,3 SUPERB FAIRYWREN GENOME DNA quality insights from de novo sequencing and assembly of the superb fairywren genome (Malurus cyaneus) 1,2,3 SEQUENCING TECH & TYPES OF DATA Yields highly fragmented assemblies. Not useful for assembly unless paired with a different technology. Illumina mate pair Illumina shotgun 250bp Jumping libraries linking across long distances across the chromosome. Illumina mate-pair Phylogenetic gap Additional resource LONG READS Long-term study Large but erroneous pieces. Error can be corrected using smaller pieces. 1x OPTICAL MAPPING Every new reference genome increases the power of broad comparative genomics, effecting novel insights into chromosome and molecular evolution. VERY large pieces but only partial pattern visible. Used to guide joining the smaller pieces together. Pieces that are close together can be found together. Order within sets unknown. What s so hard about sequencing and assembling a genome? If a genome was a jigsaw puzzle... 20x PUZZLE only 4 types of pieces large puzzle consists of smaller puzzles of different sizes > 1 large puzzle in the box with uneven copies replicate puzzles have different pieces duplicate square pieces picture on the box doesn t match what s inside 2+ near identical replicate puzzles imperfect pieces Determined ordering of some pieces. Used to guide preassembled puzzles. Helps destinguish subpuzzles. CHROMOSOME ASSEMBLY GENOME only A,C,T,G multiple chromosomes of different sizes uneven coverage across genome DNA fragmentation random repetitive elements closest reference still distant diploidy or polyplody sequencing error gap filling 30kb PBJelly ASSEMBLY STATS scaffolds contigs Illumina +PB Illumina only Size 1.01Gb 1.05Gb 0.90Gb 1.02Gb Insert sizes: 100kb - Mbs N50 6.0Mb 8.0Mb scaffolds larger than 10kb 1.01Gb genome 440 scaffolds larger than 100kb of the genome is in 153 scaffolds larger than 1Mb Illumina +PB 14Kb of the is in 0.93Gb 467Kb Great method for spanning across repetitive regions yielding quality assemblies. 10X Genomics Short reads associated to longer reads with barcodes. de novo assembly 10X Genomics Chromium scaffolding ARCS SUPERNOVA DNA Insert sizes: >30kb Great quality DNA input can yield quality de novo assemblies. Requires 30-50X coverage. Chromosome conformation information. Association between pairs of reads based on proximity in genome. DE NOVO ASSEMBLY scaffold N50 370Kb coverage contig N50 Linkage map 19x 32Kb assembly 687Mb size HiC: In vivo - robust orientation CHiCAGO: In vitro - dependent on DNA Can link scaffolds into chromosome-scale assemblies. SCAFFOLDING molecule size 46Kb stringency 280 birds Quality of assembly depends on density of markers and number of individuals. Can build linkage groups associated with chromosomes. chr1... chrz 8.3Mb Mb AFFILIATION Annotation is a beast in its own right. Proper annotation first requires RNA sequencing from a range of tissue types. The sequencing is then followed by an incredibly time-intensive computational pipeline. Although it s not ideal, quick and dirty annotation can be achieved by using gene sets from existing databases. Don t forget that repeats, not just genes, need to be annotated too! Annotation still in planning stage... chromosome identification LASTZ SEQUENCING RID HYB Input DNA? LY EMB ASS Heterozygosity? 0.7 CRIMAP FUNDING What about annotation? Budget? 8.0Mb N50 linkage analysis F9 gens Genetic markers associated with a known pedigree. Where do I start? scaffold 0.5 DaRT-seq 26K SNPs One correct solution, infinite incorrect answers, difficult to check... Genome size? of the genome is in 1, Gb Illumina only Labeling of motifs across extremely large DNA fragments. HiC / CHiCAGO Likelihood of a pair of pieces being next to each other. Helps destinguish subpuzzles. LoRDEC up to Oxford Nanopore BioNano long-read error correction 27 SMRT CELLS Requires 50x for de novo, but 20x sufficient for gap filling existing assemblies. Single molecule, long read sequence with high error rate. LINKED READS Chromosome-scale bird genomes have been unevenly sampled across the bird tree. The fairywren belongs to an unsampled oscine passerine clade, filling in a gap in reference bird genomes. ALLPATHS 7Kb 1x PacBio RSII Insert sizes: 5-30kb & improving Requires in-house optimization and high coverage for de novo assembly. Fairywrens have been studied for 30+ years in the Australian National Botanical Gardens. There is anabundance of life history and ecological data which we can link to the genomic data. 5Kb 1x Insert sizes: 5-15kb & improving The broader aim of my PhD thesis is to understand speciation processes using genomic data. This genome will serve as a reference for studying genomic introgression across a bird suture zone. scaffolding 100bp PE (HiSeq2500) insert sizes Single molecule, long read sequence with high error rate. Speciation genomics 4x 3Kb Why are you sequencing the fairywren genome? ALLPATHS 500bp 17x Used for scaffolding shotgun assemblies. Often need at least 2 different fragment sizes. PacBio de novo assembly 100 bp PE 300 bp PE HiSeq 2500 MiSeq Typical DNA insert size: 2-10kb Large jumping libraries: 10-50kb BIOINFORMATIC PIPELINE ASSEMBLY PIPELINE

22 contigs N50 scaffolds 14 Kb 6 Mb

23 contigs N50 scaffolds 14 Kb 6 Mb 467 Kb 8 Mb

24 N50 contigs scaffolds 14 Kb 6 Mb 467 Kb 8 Mb 467 Kb 10 Mb

25 N50 contigs scaffolds 14 Kb 6 Mb 467 Kb 8 Mb 467 Kb 10 Mb