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. . There is going to be a lot of bookkeeping, defensive measures taken to protect against you-know-who, The Dark Lord of False Discoveries. . Let it run in a command window and dont interrupt the process. 64 . . The cat command concatenates one or more files and stars producing them on the output stream: cat SGD_features.tab The command above will print the contents of the file to the current output stream which happens to be your terminal window. . . . . . . . . . 211 211 211 212 214 214 215 215 215 215 216 VIII GENE ONTOLOGY 29 Gene ontology 29.1 What is the Gene Ontology (GO)? . 8.1 How do I set up my computer? . . While it is a simplistic approach, it is an excellent start to creating a reusable workflow. . . . . . . . . . . . 11.8 5: Making new directories . . . cat goa_human.gaf | cut -f 2 | sort | uniq -c | sort -k1,1nr > prot_counts.txt # What are the ten most highly annotated gene names in the GO dataset? . . . It is typically the first step performed after data acquisition. . . . . 90 . Merge two table files: csvtk join -H -t 1.fa.tsv 2.fa.tsv | cat -A seq1^Iaaaaa^I^ICCCCC^I$ seq2^Iccccc^I^IGGGGG^I$ seq3^Iggggg^I^ITTTTT^I$ Step 3. . 2. . . . . . We cover both the foundations and their applications to realistic data analysis scenarios. . . Access to the Biostar Handbook website for two years. . . . . . . 161 Its best if you learn to use a proper text editor from the start. . . . . . . One free option to use as an introduction to the Biostar Handbook is the Harvard Chan Bioinformatics core training modules. Instead of going ahead with analyzing the data we have to spend precious mental resources on untangling a data annotation mess. . . The choice is to select or type yes unless otherwise instructed by the book. 697 Figure 110.1: o motif in a binding location would be interesting only if seeing that motif has predictive value. . 11.15 12: Man pages If every Unix command has so many options, you might be wondering how you find out what they are and what they do. . . 362 CHAPTER 52. 136 . . . Superficially the default operation of awk appears to split lines into columns by any whitespace (spaces, tabs) in reality, it does a lot more than that. . mkdir -p bam # Align then sort and convert to BAM file. 8.2 Is this going to be difficult? . yeastgenome.org/sequence/S288C_reference/genome_releases/. . . Using BLAST is a bit like using Google when searching for information. . . . . . . . . . . 111.2 What are the processing steps for ChIP-seq data? . MISCELLANEOUS UNIX POWER COMMANDS 117 View the penultimate (second-to-last) 10 lines of a file (by piping head and tail commands): #tail -n 20 gives the last 20 lines of the file, and piping that to head will show the tail -n 20 file.txt | head Show the lines of a file that begin with a start codon (ATG) (the matches patterns at the start of a line): grep "^ATG" file.txt Cut out the 3rd column of a tab-delimited text file and sort it to only show unique lines (i.e. . . It is also hard to shake the feeling that each data providers ultimate goal is to be too big to fail; hence, there is an eagerness to integrate more data for the sole purpose of growing larger. . . 6.2 How much computing power do we need? . . . Finally, by considering the correlated changes in gene expression, FCS methods account for dependence between genes in a pathway, which ORA does not. end up with less reliable results than focused experiments. . . . . . . . . . . The following is a FASTA file with two FASTA records: >foo ATGCC >bar other optional text could go here CCGTA As it happens, the FASTA format is not officially defined - even though it carries the majority of data information on living systems. . . . . . . . The classification produces fewer results overall only 2424 sequences were classified instead of 7424: 78272 sequences (14.88 Mbp) processed in 5.743s (817.7 Kseq/m, 155.45 Mbp/m). . . . . Most likely the word region means a wider interval, not just the reported peak coordinate. . . WHAT IS A SHELL? . . . . . . Variants that occur on the same DNA molecule form a haplotype. . First, you need to think about how the workflow should be. . . . . . . . Whatever I we were about to investigate got derailed, whats with all the Ns? . . HOW ARE GO TERMS ORGANIZED? . . 118 . Here is a better explanation. CONTENTS 5 11.3 Typeset Conventions . . . 2. . . . . . . 52.1 What part of the FASTQ file gets visualized? . . . 70 When presented a gene list like the one shown below Tmem132a Myl3 Myl4 Tnnt2 Hspb7 Tnni3 Actc1 DAVID will generate functional interpretations: 14 genes (My13, My14, Tnni3,) from this list are involved in Cardiac Muscle Contraction 4 genes (Actc1, Myh6, Myh7, Tnnt2) have the role of ATPase Activity and so on. . . 1 https://www.ncbi.nlm.nih.gov/grc/help/patches/ 153 154 17.2 CHAPTER 17. . . . . . . . . . . . . What you need is a proper track record of what you did. . . . Here are the outputs when run in 2016 2008 2009 2010 2011 2012 2013 2014 2015 2016 9682 13322 15026 23490 16428 60555 34925 33096 235077 And when run at the beginning of 2019: 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 8694 12512 13011 21186 14693 57417 23636 19656 24109 56695 190351 This result is quite surprising, and we dont quite know what to make of it. . . . . . . . . 541 global-align.sh query.fa mutated.fa on our system this produces the following (the mutations are random so you will get something different): CGGACACACAAAAAGAAAGAAGAATTTTTAGGATCTTTTGTGTGCGAATA |||||||||||||||||||||||||||||| ||||||||||||||||||| CGGACACACAAAAAGAAAGAAGAATTTTTA-GATCTTTTGTGTGCGAATA Mutate the query to contain a 2 base block mutation: cat query.fa | msbar -filter -block 3 -minimum 2 -maximum 2 > mutated.fa The alignment now shows: CGGACACACAAAAAGAAAGAAGAATTTTTAGGATCTTTTGTGTGCGAATA ||||||||||||||||||||||||||||||||||||||||||| ||||| CGGACACACAAAAAGAAAGAAGAATTTTTAGGATCTTTTGTGT--GAATA We can introduce more than one mutation with the -count parameter: cat query.fa | msbar -filter -count 2 -block 3 -minimum 2 -maximum 2 > mutated.fa one result could be: CGGACACACAAAAAGAAAGAAGAATTTTTAGGATCTTTTGTGTGCGAATA ||||||||||||| |||||||||||||||||| ||||||||||||||| CGGACACACAAAA--AAAGAAGAATTTTTAGGA--TTTTGTGTGCGAATA Since the tool selects mutations randomly, when choosing substitutions it is possible to mutate the sequence back to itself i.e. . . . . . . . . . . . . . . . . . . . . . . . 151 . . . . . . . . . . . . . 505 In the future examples we will combine the two commands into one. . . . Remember that the real genome does not need to match the reference - real, biological changes could look like incorrect mapping. . . . . . . . echo "Call me Ishmael." Since it is a process that alters the data, we must be extremely cautious not to introduce new features into it inadvertently. . 52.8 What does the sequence length histogram show? . . . . . 91.6 How do I extract variants excluding a specific region? . . . . 52.4 How does FastQC work? . . . Which fragments will be considered: The apparent fragment size (aka TLEN from SAM?) . . The main problem with wc -l SGD_features.tab that it makes the process less reusable if you wanted to run another tool with the same file youd modify the beginning of the command, whereas when using cat you can delete the last command and add a new one. 21.3 What is RefSeq? . . . . Stop QC if the quality appears to be satisfactory. . . 17.1 How many genomic builds does the human genome have? . Previously I have mentioned how gene was the most misused word in biology. . . . There is no universal rule, method or protocol that would always produce correct answers or guarantee some range of optimality. . 228 . . . . . . . . . . Alas the colormath library installed with conda is a prior version and not the latest that includes the fixes. 7.3 What are the rules of a bioinformatics analysis? . . . . . 173 . . . . . Corollary: An expert scientist can unwittingly publish a Nature publication with tiny p-values, impressive and compelling visualizations even when the underlying data is not different from random noise1 . . 11.9 6: Getting from A to B . . . You can also automate the process to generate the names at the command line. . . . . Long story short, dont put too much stock into p-values and other measures. . . . . 137 Figure 14.2 Everythings gonna be alright Everythings gonna be okay Its gonna be a good, good, life Thats what my therapists say Here is what is happening. . 107.7Do peaks represent binding? . . Here is an example: Name ERCC-00130 ERCC-00092 ERCC-00083 ERCC-00096 ERCC-00060 ERCC-00117 ERCC-00002 ERCC-00079 ERCC-00061 1 Mix1 30000 234 0.028 15000 234 0.057 15000 58 0.057 Mix2 7500 58 0.007 15000 234 0.057 30000 117 0.114 M1/M2 4 4 4 1 1 1 0.5 0.5 0.5 log2(M1/M2) 2 2 2 0 0 0 -1 -1 -1 http://data.biostarhandbook.com/rnaseq/ERCC/ERCC-information.pdf 630 100.2. . . Sequence format and type are automatically detected. . . There is variability and errors in sample extraction, handling and of the lenghty process of analysis. . . . . The history of estimating the magnitude of gene expression is short and convoluted. 27.2 Are there other ontologies? . A log2 fold change of 1 means a doubling of the expression level, a log2 fold change of -1 shows show a halving of the expression level. . You can also always change to a directory based on its absolute location. . . Again one of the differences between aligners. A particular variant of it, the clustered heatmap is so commonly used and so useful (yet non-trivial) to obtain that weve included a script that will generate one for you. . 24.2 How to produce an overview of FASTQ files? Minimally we recommend the following: # # A minimal BASH profile. . . . For example: the - character may indicate a gap (space), the | character is used to display a match, 426 CHAPTER 65. 54.4 Why do we need to trim adapters? . The word reference implies that we are comparing against a ground truth but it is the other way around. . . . . pwd ls Simple Unix tools let us answer a wide variety of questions. . . . ARE FASTQ ERRORS VALUES ACCURATE? Access your terminal. . . . The input consists of a list of genes. . . It has many nice features as well and the documentaion is also very good. cat SGD_features.tab | cut -f 2 | grep ORF | wc -l 12.3.17 Can I select multiple columns? local-align.sh THISLINE ISALIGNED -data BLOSUM90 Using the BLOSUM90 scoring scheme produces a much longer alignment: SLI-NE :|| || ALIGNE 66.4. . . Variant annotation means predicting the effects of genetic variants (SNPs, insertions, deletions, copy number variations (CNV) or structural variations (SV)) on the function of genes, transcripts, and protein sequence, as well as regulatory regions. At the same time, you have to realize that you can only succeed at changing how you think when you perform these actions. . 6.1 What is the recommended computer for bioinformatics? . . . You will have quite a hard time following the analysis as the processes used to generate the results will often rely on seemingly subjective decisions. A bgzip file can be decompressed with gzip but only the specialized bgzip command can create a file in the Blocked GNU Zip Format file. . . 7.6 What does simple mean? . 16.8 Is there a list of all resources? . . . The general processing steps are as following: 1 https://github.com/crazyhottommy https://github.com/crazyhottommy 3 http://crazyhottommy.blogspot.hu/ 2 705 706 CHAPTER 111. . . . . . 3. Over this time it was unclear to users whether the data that the server operates on includes the most up to date information. . . . . Converting semi-ambiguous IUB codes to N. . Subject: the sequence entry in the target that produced an alignment. . . . . . . Early warning: when visiting bioinformatics data sites, you are likely to be subjected to an information overload coupled with severe usability problems. . . Whereas alignments or counting overlaps are mathematically well-defined concepts, the goals of a typical experiment are more complicated. . But then few things are more wasteful and frustrating than having to redo something just because we forgot a seemingly essential step. . . . . . . 57.12How can I assign default values to a variable? . . . . . . For example, select alignments that overlap with coordinate 323,567,334 on chromosome 2 Quick selection and filtering of reads based on attributes. . . . . . 695 removing some (or many) of the resulting peaks. . . . . . . . . 257 . Typically, a sole genius is behind each, an individual with uncommon and extraordinary programming skill, a person that has set out to solve a problem that is important to them. . . NR_118889.1 NR_118899.1 NR_074334.1 NR_118873.1 NR_119237.1 NR_118890.1 NR_044838.1 NR_118908.1 NR_118900.1 1300 1367 1492 1492 1492 1331 1454 1343 1376 36819 1658 224325 224325 224325 1816 1381 1068978 1655 Amycolatopsis azurea Actinomyces bovis Archaeoglobus fulgidus DSM 4304 Archaeoglobus fulgidus DSM 4304 Archaeoglobus fulgidus DSM 4304 Actinokineospora fastidiosa Atopobium minutum Amycolatopsis methanolica 239 Actinomyces naeslundii 458 CHAPTER 70. . . . . With time you will immediately be able to recognize the format from its extension or 16.1 A Quick look at the GENBANK format. . Whereas RPKM refers to reads, FPKM computes the same values over read pair fragments. . . . Ion/Oxford Nanopore technologies are not very robust when homopolymers (e.g., AAAAA) of significant length are present in samples. . . . These will finish much faster. . . 9.4 Which text editor to choose? . 182 23.2 How to recognize FASTQ qualities by eye . . . . 94 For that and other reasons, the majority of life scientists do not make use of the SO relationships and use the SO terms only. . . . . . For example the following is an alignment between two sequences ATGCAAATGACAAATCGA and ATGCTGATAACTGCGA : ATGCAAATGACAAAT-CGA |||| |||.||.| ||| ATGC---TGATAACTGCGA Above 13 bases are the same (13 identities), 5 bases are missing (5 gaps), 2 bases are different (2 mismatches). . . Convert sam to sorted bam: # avoid writing unsorted bam to disk # note that samtools sort command changed invoke pattern after version 1.3 https://www. . . . . . . . . . 55 55 55 56 56 56 57 58 58 59 59 60 6 How is bioinformatics practiced? . . . . . . . . . 54.3 Can we customize the adapter detection? If we mark the first fragment as = then we can see that depending on the DNA fragment lengths and our measurement (read) lengths the measured reads could fall into three different orientations. items: 4 . . . . # Copyright (c) 1966-2016 Johns Hopkins University. . . . In general, all short read aligners operate on the same principles: 1. . The requirement to have a particular kind of information in the BAM file may also be a significant factor. . . . . . . . . An alternative tutorial is available online at https://github.com/griffithlab/rnaseq_ tutorial/wiki Note: We have greatly simplified the data naming and organization and processing. . There are several methods to compare replicates and estimate the magnitude of changes. . For that purpose, we list both files on the command line: bwa mem $REF $R1 $R2 > bwa.sam Note how the same data can be aligned in either single end or paired end mode. . . Several competing methods have been proposed to account for the problems that we have enumerated above. . . . . . . . . . . . . 57.11. . . . . If you activate the quality filter at the start of a run, the base-called data will get stored into pass or fail subfolders in the specified download folder. 59.7 How can I build more complex conditions in Awk? . cat runinfo.csv | grep "454 GS FLX Titanium" | cut -f 1 -d , > srr-16s.txt # Store the 16S data here. . . For some types of data, it does not matter at all what plan you pick. . . 2.0.3 What are the licensing terms? . ermineJ6 Standalone tool with easy to use interface. It can be best learned via an interactive service like https://regexone.com/ or many others where the pattern and its effect are instantly visualized. . . 78 79 79 80 80 80 81 82 82 83 83 84 84 84 84 85 . . . From early on NCBI offered a web API interface called Entrez E-utils1 then later released a toolset called Entrez Direct2 . TYPESET CONVENTIONS 99 Figure 11.1 11.3 Typeset Conventions Command-line examples that you are meant to type into a terminal window will be shown indented in a constant-width font, e.g. . . . . . Michael Browner 117.3How do I set up the BLAST for taxonomy operations? But there is a downside to all this power - as our knowledge grows the possible rate of false discovery increases faster than what most scientists assume. . . 42 E.g. . . . . So now that you know what bioinformatics is all about, youre probably wondering what its like to practice it day-in-day-out as a bioinformatician. . character allows you to rerun a previous command. . ahead and reach us at . . . 2. . . # one has to run ROSE inside the ROSE folder. 71.5 What is read mapping? . . . . By contrast, studies using ChIP-Seq data would be essentially mapping-oriented. 603 604 95.4 CHAPTER 95. Whereas both lower-case and upper-case letters are allowed by the specification, the different capitalization may carry additional meaning and some tools and methods will (tacitly again) operate differently when encountering upper- or lower-case letters. . . For example for a diploid organism the GT field indicates the two alleles carried by the sample: 0/0 0/1 1/2 1/1 2/2 - the sample is a homozygous reference the sample is heterozygous, carrying one of each the REF and ALT alleles would indicate a heterozygous carrying one copy of each of the ALT alleles. . . . . . . . . . . . . . . Using Word to edit text will eventually cause (devious) errors. . . 11.1310: Finding your way back home . . . . You will not get spatial information about fragments unless you choose to do paired-end sequencing. . . . . . . . 90.6 What are VCF records? . . wget -nc http://data.biostarhandbook.com/sra/sra-runinfo.sh # Make the script executable chmod +x sra-runinfo.sh # Run on each pair, but just request in parallel at a time so the NCBI does not ban you. . . . . . . . . 96.4 What types of statistical tests are common? . . WHAT IS DATA? . . . The format has a hierarchical structure with groups for organizing data objects and datasets which contain a multidimensional array of data elements. . . . . . . . WHICH PROGRAMMING LANGUAGE SHOULD I LEARN? . . . . . The more unknowns about the genome under study, the more critical it is to correct any errors. . Commas, on the other hand, are commonly used in different contexts as well. . . . . . . UNDERSTAND YOUR DATA UHR + ERCC Mix1, Replicate 1, UHR_1 UHR + ERCC Mix1, Replicate 2, UHR_2 UHR + ERCC Mix1, Replicate 3, UHR_3 HBR + ERCC Mix2, Replicate 1, HBR_1 HBR + ERCC Mix2, Replicate 2, HBR_2 HBR + ERCC Mix2, Replicate 3, HBR_3 99.3 How do I download the example data? . . . . . A protein bound to a DNA molecule can protect it from being washed away - then in a second pulldown stage, only the DNA bound to a particular protein is kept. . . . . . . First, peaks are found just like any other ChIP-Seq data set. . 302 41.3 Will the best bioinformaticians on the planet produce reproducible analyses? . .