Introduction to Next Generation Sequencing
Francisco J. (Paco) Ruiz-Ruano
Universidad de Granada
Botucatu, September 2016
Parte I
Introduction to NGS technologies and Linux
FJ Ruiz-Ruano Introduction to NGS 2 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencing
Parte II
Genomics
FJ Ruiz-Ruano Introduction to NGS 3 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencing
1 Genome assembly
2 Repeated elements assembly
3 Abundance and divergence
4 Amplicon sequencing
FJ Ruiz-Ruano Introduction to NGS 4 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencing
1 Genome assembly
2 Repeated elements assembly
3 Abundance and divergence
4 Amplicon sequencing
FJ Ruiz-Ruano Introduction to NGS 5 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencing
FJ Ruiz-Ruano Introduction to NGS 6 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencing
FJ Ruiz-Ruano Introduction to NGS 7 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencing
FJ Ruiz-Ruano Introduction to NGS 8 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencingGenome De novo Reference Satellite
1 Genome assembly
2 Repeated elements assemblyGenomeDe novoReferenceSatellite
3 Abundance and divergence
4 Amplicon sequencing
FJ Ruiz-Ruano Introduction to NGS 9 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencingGenome De novo Reference Satellite
Using an assembled genome
We search in NCBI Genome
We assembly and annotated with RepeatModeler
FASTA file as output useful for annotation
FJ Ruiz-Ruano Introduction to NGS 10 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencingGenome De novo Reference Satellite
De novo assembly with RepeatExplorer
http://www.repeatexplorer.org/
FJ Ruiz-Ruano Introduction to NGS 11 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencingGenome De novo Reference Satellite
http://www.repeatexplorer.org/
FJ Ruiz-Ruano Introduction to NGS 12 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencingGenome De novo Reference Satellite
http://www.repeatexplorer.org/
FJ Ruiz-Ruano Introduction to NGS 13 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencingGenome De novo Reference Satellite
Repeat Explorer options
We select about 2x200000 reads
We use de paired end mode to check connections
We can compare abundances between several libraries
We can use a custom database of repeated elements
FJ Ruiz-Ruano Introduction to NGS 14 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencingGenome De novo Reference Satellite
Repeated elements using a reference
MITObim maps reads to a reference and performs an iterativeassembly
A custom script can select reads to the reference with BLAT andwe can manually assembly it with CAP3 or Newbler. We can alsouse RepeatExplorer.
FJ Ruiz-Ruano Introduction to NGS 15 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencingGenome De novo Reference Satellite
satMiner: DNA satellite assembly
Clustering Filtering
1
100
200
1 100 200
satDNAselection
Iteration
rexp_prepare.py +RepeatExplorer
rexp_select_contigs.py+ run_deconseq.py
FASTQFASTQ
IntragenomicVariation
97.5
41.6
38.4
31.6
100
39.5
42.7
37.9
82.8
32.9
80.5
64.3
62.8
69.5
66.661.9
59
1110:13839:4083
1114:20149:71475
1105:6342:11438
1113:18
549:7
6919
1112:4
696:77
530
1109:75
35:1611
0
1101:6884:33932
1205:2026:
164651103:18827:22199
1113:16093:670821104:9532:67053
1116:4300:86873
1115:13211:43258
1108:18251:98208
1114:4754:7598
1114:67
59:56
259
1110:136
12:67877
1105:10786:86782
1115:10566:93694
1102:5733:39467
1106:8492:512321105:6670:9536
1115:15184:69666
1205:13273:96513
1108:4109:8616
1104:6629:58775
1107:5025:43656
1104:16313:54396
1201:19562:94344
0.07
Sat-16
Sat-12
Homologyrm_homology.py
Abundanceand divergence
Unfiltered raw reads satDNA
consensus
SatDNA Analysis
SatDNAMining
RepeatMasker
rm_getseq.py
SouthNorth
LmiSat37A-238
LmiSat26A-240
LmiSat51A-241
72s,2id(2)
12s
76s,7id(15)
LmiSat26A-240
1 50 100 150 200 251
FJ Ruiz-Ruano Introduction to NGS 16 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencingGenome De novo Reference Satellite
DNA satellite assembly
Iterative assembly with RepeatExplorer and filtering with DeconSeq
Additional analysis: Homology, abundance and divergence,Intragenomic diversity
https://github.com/fjruizruano/satminerhttp://www.nature.com/articles/srep28333
FJ Ruiz-Ruano Introduction to NGS 17 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencing
1 Genome assembly
2 Repeated elements assembly
3 Abundance and divergence
4 Amplicon sequencing
FJ Ruiz-Ruano Introduction to NGS 18 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencing
Repeat Landscape with RepeatMasker
a b
http://link.springer.com/article/10.1007/s00412-016-0611-8
FJ Ruiz-Ruano Introduction to NGS 19 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencing
Coverage with SSAHA2
ORF5'-UTR
3'-UTREmoSat27-102
0B +B0B +B
http://link.springer.com/article/10.1007/s00412-016-0611-8
FJ Ruiz-Ruano Introduction to NGS 20 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencing
1 Genome assembly
2 Repeated elements assembly
3 Abundance and divergence
4 Amplicon sequencing
FJ Ruiz-Ruano Introduction to NGS 21 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencing
FJ Ruiz-Ruano Introduction to NGS 22 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencing
FJ Ruiz-Ruano Introduction to NGS 23 / 24
Genome assembly Repeated elements assembly Abundance and divergence Amplicon sequencing
FJ Ruiz-Ruano Introduction to NGS 24 / 24