A talk I gave at the Dec 2013 Assembly Masterclass at UC Davis. Really licensed under CC0. UPDATED May 2014, for the presentation I gave at the combined SeRC Nordic Assembly Workshop in Stockholm, Sweden, May 14th 2014

1. Assembly – before and after
Lex Nederbragt
lex.nederbragt@ibv.uio.no
@lexnederbragt

2. A warning
The list is by no means complete
Nor do we have experience with all the programs mentioned

3. Sample DNA Reads
Genome
assembly
Sequencing AssemblyDNA isolation
QC QCQC

4. Reads
Genome
assembly
Assembly
QC

5. Fastqc

6. Prinseq

7. Many others…
www.nipgr.res.in/ngsqctoolkit.html

8. preqc (sga)
http://arxiv.org/abs/1307.8026

9. Reads
Genome
assembly
Assembly
Grooming

10. Format conversion
http://en.wikipedia.org/wiki/FASTQ_format
Fastq format hell

11. Adapter/quality trimming
http://www.biostars.org/p/53528/
Celera assembler
Overlap based trimming
Fastx Toolkit
Seqtk
PrinSeq
NGS QC Toolkit
Trimmomatic
BioPieces
Cutadapt
…
…

12. Mate pair splitting and orientation
150 – 600 bases
Illumina paired end reads
2 – 40 kilobases
Illumina mate pair reads
2 – 40 kilobases
454 mate pair reads
linker

13. Mate pair splitting and orientation
Illumina paired end reads
Illumina mate pair reads
454 mate pair reads
linker
junctionjunction
+ +
paired end reads
‘contamination’

14. Mate pair splitting and orientation
Illumina paired end reads
Illumina mate pair reads
454 mate pair reads
linker
junctionjunction
+ +
paired end reads
‘contamination’
Check what orientation
your assembler expects
for the reads!

15. Reads
Genome
assembly
Assembly
Preparing

16. Error-correction
Stand-alone or built into assembler

17. Merging pairs
List from Torsten Seeman’s blog
http://thegenomefactory.blogspot.no/2012/11/tools-to-merge-overlapping-paired-end.html
COPE http://sourceforge.net/projects/coperead/
SeqPrep https://github.com/jstjohn/SeqPrep
FLASH http://www.cbcb.umd.edu/software/flash
fastq-join http://code.google.com/p/ea-utils/wiki/FastqJoin
PANDAseq https://github.com/neufeld/pandaseq
mergePairs.py http://code.google.com/p/standardized-velvet-assembly-report/source/browse/trunk/mergePairs.py
Recent addition

18. Extend reads
http://140.116.235.124/~tliu/arf-pe/

19. Digital normalisation
http://arxiv.org/abs/1203.4802

20. Estimate kmer to use
preqc (SGA)
http://arxiv.org/abs/1307.8026

21. Reads
Genome
assembly
Assembly
What can the reads tell us about the genome

22. kmer-based
preqc (SGA)
Kmerspectrumanalyzer
http://arxiv.org/abs/1307.8026
Khmer from Titus

23. Reads
Genome
assembly
Assembly
This
talk

24. Reads
Genome
assembly
Assembly
QC

25. Genome
assembly
Comparing to each other
Metrics
Merging
Improvement
Visualization
Validation
Comparing to reference

26. Genome
assembly
Comparing to each other
Metrics
Merging
Improvement
Visualization
Validation
Comparing to reference

27. Assemblathon stats
http://korflab.ucdavis.edu/datasets/Assemblathon/Assemblathon2/Basic_metrics/assembla
thon_stats.pl
OR
https://github.com/lexnederbragt/sequencetools/

28. Genome
assembly
Comparing to each other
Metrics
Merging
Improvement
Visualization
Validation
Comparing to reference

29. Gap closing
IMAGE2

30. Correcting bases
Quiver from Pacific Biosciences

31. Separate scaffolding

32. Genome
assembly
Comparing to each other
Metrics
Merging
Improvement
Visualization
Validation
Comparing to reference

33. Assembly merging/reconciliation

34. Genome
assembly
Comparing to each other
Metrics
Merging
Improvement
Visualization
Validation
Comparing to reference

35. Mapped genomic reads
FRCBAM

36. Mapped transcriptomic reads

37. Gene finding

38. Binning
Bacteroides
Proteobacteria
Cyanobacteria
Per-con g read depth
Nederbragt et al, 2010

39. Genome
assembly
Comparing to each other
Metrics
Merging
Improvement
Visualization
Validation
Comparing to reference

40. Genome browser(s)
IGV

41. Genome
assembly
Comparing to each other
Metrics
Merging
Improvement
Visualization
Validation
Comparing to reference

42. Comparative measures
Log Average Probability (LAP)
Assembly Likelihood Evaluation (ALE)
See also Howison, Zapata2 and Dunn (2013) Toward a
statistically explicit understanding of de novo sequence
assembly doi: 10.1093/bioinformatics/btt525

43. Genome
assembly
Comparing to each other
Metrics
Merging
Improvement
Visualization
Validation
Comparing to reference

44. Reference comparison
Mauve assembly metrics

45. Review

46. Too many tools…
http://seqanswers.com/wiki/Software/list

47. Too many tools…
http://wwwdev.ebi.ac.uk/fg/hts_mappers
88 short-read mappers

48. Embargo!

49. Benchmarking, anyone?

50. All-in-one assembly pipeline
doi:10.1186/1471-2105-15-126