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