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Introduction to NGS Fotis E. Psomopoulos CODATA-RDA Advanced - PowerPoint PPT Presentation

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Introduction to NGS Fotis E. Psomopoulos CODATA-RDA Advanced Bioinformatics Workshop, 19-23 August 2019, Trieste, Italy Sequencing Technology 2 Introduction to NGS Tuesday, August 20th 2019 Changes and Timing past decade 3 Introduction to

  1. Introduction to NGS Fotis E. Psomopoulos CODATA-RDA Advanced Bioinformatics Workshop, 19-23 August 2019, Trieste, Italy

  2. Sequencing Technology 2 Introduction to NGS Tuesday, August 20th 2019

  3. Changes and Timing past decade 3 Introduction to NGS Tuesday, August 20th 2019

  4. The (new) flow of information 4  The trinity of human, data and computer *  Extremely high bandwidth between computer and data. Human  Narrow communication channels between human and computer / data. Computer Data * http://www.kdnuggets.com/2016/08/data-science-challenges.html Introduction to NGS Tuesday, August 20th 2019

  5. Overview of costs (past, present and 5 near future) Introduction to NGS Tuesday, August 20th 2019

  6. Steps in sequencing experiments 6 Introduction to NGS Tuesday, August 20th 2019

  7. NGS analysis workflow 7 Introduction to NGS Tuesday, August 20th 2019

  8. The three stages of NGS data analysis 8  We will try to provide an overview of all steps in this course Introduction to NGS Tuesday, August 20th 2019

  9. NGS Applications are sequencing 9 applications  Whole Genome Sequencing  Gene Regulation  Epigenetic Changes  Metagenomics  Paleogenomics  Transcriptome Analysis  Resequencing  …. Introduction to NGS Tuesday, August 20th 2019

  10. End-to-end computational workflows

  11. Why QC and preprocessing 11  Sequencer output  Reads + quality  Natural questions  Is the quality of my sequenced data ok?  If something is wrong, can I fix it?  Problem: HUGE files Introduction to NGS Tuesday, August 20th 2019

  12. Sequencing Data Formats 12 Introduction to NGS Tuesday, August 20th 2019

  13. Quality before content 13 Introduction to NGS Tuesday, August 20th 2019

  14. What is quality? 14 Introduction to NGS Tuesday, August 20th 2019

  15. Trace File (high quality) 15 Introduction to NGS Tuesday, August 20th 2019

  16. Trace File (Medium Quality) 16 Introduction to NGS Tuesday, August 20th 2019

  17. Trace File (Low Quality) 17 Introduction to NGS Tuesday, August 20th 2019

  18. Phred Quality Scores 18  Phred is a program that assigns a quality score to each base in a sequence. These scores can then be used to trim bad data from the reads, and to determine how good an overlap actually is  Phred scores are logarithmically related to the probability of an error:  a score of 10 means 10% error probability,  20 means a 1% chance,  30 means a 0.1 chance, etc  A score of 30 is usually considered the minimum acceptable score. Introduction to NGS Tuesday, August 20th 2019

  19. FASTQ File Format 19  Each read is represented by four lines: 1. @ followed by read ID 2. Sequence 3. + optionally followed by repeated read ID 4. Quality line  Same length as sequence  Each character encodes the quality of the respective base Introduction to NGS Tuesday, August 20th 2019

  20. FASTQC 20  As the name implies, FastQC is way to quickly see some summary statistics to check the quality of your NGS run.  It runs both as a GUI (requires Java) and as a command line program.  Provides several statistics:  Per Sequence Quality  Per sequence quality scores  Per base sequence and GC content  Per Sequence GC Content  etc.. Introduction to NGS Tuesday, August 20th 2019

  21. Trimming 21  Knowing quality → Act accordingly  Adapter trimming  May increase mapping rates  Absolutely essential for small RNA Probably Improves de novo assemblies  Quality trimming  May increase mapping rates  May also lead to loss of information  Lots of software:  Cutadapt, Trim Galore!, PRINSEQ, etc. Introduction to NGS Tuesday, August 20th 2019

  22. Mapped Reads 22  Mapping: “align” these raw reads to a reference genome  Single-end or paired-end data?  How would you align a short read to the reference?  Old-school: Smith-Waterman, BLAST, BLAT,…  Now: mapping tools for short reads that use intelligent indexing and allow mismatches Introduction to NGS Tuesday, August 20th 2019

  23. Short read applications 23  Genotyping  RNA-Seq, ChIP-Seq, Methyl-Seq,… Introduction to NGS Tuesday, August 20th 2019

  24. Defining the question 24  Given a reference and a set of reads, report at least one “good” local alignment for each read, if one exists  Approximate answer to question: where in genome did read originate  What is “good”? For now we concentrate on:  Fewer mismatches = better  Failing to align a low-quality base is better than failing to align a high-quality base Introduction to NGS Tuesday, August 20th 2019

  25. Interlude 25 (not only) NGS File Formats Introduction to NGS Tuesday, August 20th 2019

  26. The S equence A lignment/ M ap Format 26  Generic alignment format  Supports short and long reads  Supports different sequencing platforms  Flexible in style, compact in size, computationally efficient to access  SAM File Format  BAM is the binary version of the SAM file; not human readable but indexed for fast access for other tools / visualization / … Introduction to NGS Tuesday, August 20th 2019

  27. SAM Fields 27 Introduction to NGS Tuesday, August 20th 2019

  28. Other useful formats in NGS 28  B rowser E xtensible D ata (location / annotation / scores).  used for mapping / annotation / peak locations  extension: bigBED (binary)  BEDGraph files (location, combined with score)  used to represent peak scores  Introduction to NGS Tuesday, August 20th 2019

  29. Other useful formats in NGS 29  WIG files (location / annotation / scores): wiggle  used for visualization or to summarize data, in most cases count data or normalized count data (RPKM)  extension: BigWig – binary versions, often used in GEO for ChIP-seq peaks Introduction to NGS Tuesday, August 20th 2019

  30. Other useful formats in NGS 30  G eneral F eature F ormat  used for annotation of genetic / genomic features, such as all coding genes in Ensembl  often used in downstream analysis to assign annotation to regions/peaks/…. Introduction to NGS Tuesday, August 20th 2019

  31. Other useful formats in NGS 31  V ariant C all F ormat  used for SNP representation Introduction to NGS Tuesday, August 20th 2019

  32. aaaand back to the story 32 Introduction to NGS Tuesday, August 20th 2019

  33. Mappers 33  BowTie2 is the most commonly used aligner  Employs an indexing algorithm that can trade flexibility between memory usage and running time  BWA (mem / aln) is an efficient mapper that is extensively used in RNA- Seq  STAR aligner, is an general, all-purpose aligner Introduction to NGS Tuesday, August 20th 2019

  34. HiSat2 34  Stands for:  hierarchical indexing for spliced alignment of transcripts  HISAT2 is a fast and sensitive alignment program for mapping next- generation sequencing reads (both DNA and RNA) to a population of human genomes (as well as to a single reference genome).  HISAT2 searches for up to N distinct, primary alignments for each read  Very fast  Low memory requirements Introduction to NGS Tuesday, August 20th 2019

  35. We’ve aligned the data. Then what? 35  Depending on the target study. Treatment 2 Gene Treatment 1 1 14 18 10 47 13 24 2 10 3 15 1 11 5 3 1 0 10 80 21 34 4 0 0 0 0 2 0 5 4 3 3 5 33 29 . . . . . . . . . . . . . . . . . . . . . 53256 47 29 11 71 278 339 Total 22,910,173 30,701,031 18,897,029 20,546,299 28,491,272 27,082,148 Introduction to NGS Tuesday, August 20th 2019

  36. Differential Expression 36  To determine if gene 1 is DE, we would like to know whether the proportion of reads aligning to gene 1 tends to be different for experimental units that received treatment 1 than for experimental units that received treatment 2 14 out of 22,910,173 47 out of 20,546,299 18 out of 30,701,031 vs. 13 out of 28,491,272 10 out of 18,897,029 24 out of 27,082,148 Introduction to NGS Tuesday, August 20th 2019

  37. 37 How about we try these now? Introduction to NGS Tuesday, August 20th 2019

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