Implementation and Evaluation of 10X Genomics Chromium technology
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1 Implementation and Evaluation of 10X Genomics Chromium technology Claire Kuchly & Olivier Bouchez 1
2 Chromium evaluation: pilot phase Platform installed in november 2016 Training: november 15th pilot projects: Rabbit, Tomato & Fish 2
3 Chromium 10XGENOMICS Library preparation for Illumina sequencing Long range genomics (>50 kb), haplotyping/genome phasing, structural variants detection, de novo sequencing Single cell analysis (500 >10000) Exome sequencing HiSeq3000 NovaSeq Chromium 3
4 Chromium 10XGENOMICS How does it work? 4
5 Chromium 10XGENOMICS How does it work? Complete wetlab workflow: 2 days GEM (Gel Bead in Emulsion) 4M Barcodes Input: ~1 ng HMW gdna Sequencing on HiSeq3000 2x150pb 5
6 Advantage of linked reads: genome phasing Resolve the Genome Into Multi Megabase Phase Blocks Phase the full spectrum of variants (SNVs, indels, and large scale structural rearrangements) into ultra long multimegabase phase blocks, enabling a full understanding of diploid genome sequence without the need for a reference. 6
7 Haplotyping using Chromium Results via Loupe Linked reads and structural variant calling 7
8 Single cell analysis 8
9 Chromium limits for genome sequencing Development on human genome (3 Gb), usefull for other genomes? Genome size 100 Mb minimum DNA size 50 kb minimum, 100 kb for de novo assemblies Improvments to develop for smaller genomes 9
10 Haplotyping using Chromium DNA prep, without BluePippin Young rabbit = low amount of DNA Molecular weight assessed by Fragment Analyzer 10
11 Haplotyping using Chromium Results via Loupe software Without BluePippin sizing 11
12 Haplotyping using Chromium DNA prep Rabbit DNA from 2 parents & 2 babies Genome ~ 2 Gb Extraction by MagAttract (Qiagen) from blood, molecular weight assessed by Fragment Analyzer 1,13 µg (40% >40 kb) 68 ng 12
13 Haplotyping using Chromium With BluePippin sizing : Results via Loupe software With BluePippin sizing Input DNA size improvment = longer phase block 13
14 DNA QC: New Femto Pulse Same DNA QC on Fragment Analyzer and Femto Pulse => better resolution 14
15 10X Applications 15
16 Our 10X Applications 16
17 Genome & Exome solution Linked Reads analysis software 17
18 LongRanger software Analysis pipelines that perform : Sample demultiplexing ( with Illumina software ) Barcode processing Alignment ( Lariat ) Quality control Variant calling Phasing Structural variant calling Software with few parameters for the analysis Not easy to be run on our cluster 18
19 Sequencing Quality Control Quality of input DNA 19
20 Differents views Haplotype 1 Haplotype 2 No Phasing Phasing view Indels, SNVs, SVs (50bp to up 30Kb) Structural variant view deletion, insertion 20
21 Our 10X Applications 21
22 De Novo Assembly solution 22
23 Genomes supported for assembly Genomes Supported Human germline genomes Mamalian genomes High repeat content Small genomes (100 Mb or greater) Genomes having ploidy > 2 Microbes (<100 Mb) Human Non Germline (e.g Cancer) Large genomes (>3.2 Gb) Work well May work but may increase run time Unlikely to work well or risky 23
24 DNA Size recommendation DNA size is the key point for a successfull analysis Supernova Analysis Long Ranger Analysis 80 kb 60 kb 40 kb 20 kb 24
25 Examples Olivier Bouchez & Claire Kuchly 25
26 10X for Small Genomes? Genome size 400Mb 38X cov > 17% fragments Length N50 L50 Contig number 38X Cov 388,8 Mb 303, ,913 56X Cov 389,3 Mb 2,104, , X 56X Cov + linked 446,4 389,3 Mb 2,550,395 3,646, ,797 24,075 reads 100X Cov 446,4 Mb 2,550, ,797 56X cov > 24% fragments 1 lane HiSeq3000 ~200X cov 100X cov > 50% fragments 100X Cov + linked reads 446,4 Mb 2,550, ,694 Good assembly for a small genome with 10X data only High number of contigs despite a great L50 and N50 All linked reads 26
27 PacBio RSII + 10X Chromium =? For assembly purposes, are PacBio RSII and 10X Chromium results similar or is there a benefit to use them together? (~ 1Gb) VS 27
28 PacBio RSII + 10X Chromium =? For assembly purposes, are PacBio RSII and 10X Chromium results similar or is there a benefit to use them together? (~ 1Gb) + With the information of the linked reads, we can improve the primary assembly and make an scaffold assembly 28
29 Thanks! Jerôme Gouzy Laboratoire de Physiologie et génomique des poissons Yann Guiguen Christophe Klopp NGS team: Olivier Bouchez PacBio team: Alain Roulet Céline Roques 10X Genomics team: Adeline Chaubet Sophie Valière Pauline Heuillard Frédéric Martins Bioinfo team: Maxime Manno Anaïs Poiraudeau Claire Kuchly Céline Vandecasteele 29
30 Stats assemblage 10X supernova 30
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