. . . It is best if these are installed right away. . . . . . . . . So youd have to apply some downsampling but without knowing beforehand where each measurement belongs. 5. . 2. Yes there is. Otherwise, youll end up with a mixed data of overlapped single-end reads, and non-overlapping paired-end reads - only adding to the complexity of the subsequent tasks. . . . . . 130 CHAPTER 13. When the sequencing data looks terrible from the beginning, it is best to move on and collect new data. . 1%) in which a single nucleotide - A, T, C or G of a shared sequence differs between members of a biological species (or paired chromosome of the same individual). . 95.12Do I always need an advanced statistical analysis? . . . . . . Sequencing occurs by measuring changes in the ionic current as DNA translocates through protein nanopores in an insulated membrane on the flow cell. . . . . . . HOW DO I EXTRACT PAIRED READS FROM TWO PAIRED-END READS FILES?193 cat changes.tsv Hypothetical putative hypothetical putative Putative putative Overview the FASTA headers containing hypothetical: seqkit grep --by-name --use-regexp --ignore-case --pattern hypothetical viral.1.protein.faa gi|526245016|ref|YP_008320342.1| hypothetical protein IBBPl23_06 [Paenibacillus phage phiI gi|526245019|ref|YP_008320345.1| hypothetical protein IBBPl23_09 [Paenibacillus phage phiI gi|526245020|ref|YP_008320346.1| hypothetical protein IBBPl23_10 [Paenibacillus phage phiI A regular expression, ([ ]+ )(\w+), was used to specify the key to be replaced, which is the first word after the sequence ID in this case. . . . . GZIP extension .gz, program names are gzip/gunzip. . . 11.1815: Creating empty files with the touch command 11.1916: Moving files . . . 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)? . . A more appropriate distance normalization should divide with a value that accounts for the potential changes of the total transcript length T. gene expression = N / L * 1 / T A way to incorporate both the number of counts and the length into T is to sum the rates: T = sum Ni/Li where i goes over all observed transcripts and Ni are the reads mapped to a transcript of length Li. . . . . you want to specify mouse genome for mapping reads. . . . . . . BLAST: BASIC LOCAL ALIGNMENT SEARCH TOOL 68.4 Can BLAST be used for pairwise alignments? . . . . . . . . . . This measured read has a length associated with it, but this length has nothing to do with the original fragment length. . Welcome to the Biostar Handbook; Over the author; Enigma bioinformatics? . . . . . . The update might have been in response to the growing chorus of complaints: 5 https://david.ncifcrf.gov/home.jsp 238 CHAPTER 31. . . . . . 6.4 What about the cloud? . 106 CHAPTER 11. 94.5.2 Classifying against a transcriptome When classifying against a transcriptome the input data will be: 1 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078644 596 CHAPTER 94. . perhaps that was the plan all along, in which case I must admit, the FLAG is not such a bad thing after all. . Awk has special patterns called BEGIN and END to run specific tasks only once. . . . . . . . . . . . . . Careful when using file redirection (>), it will overwrite any existing file of the same name When you are using more or less, you can bring up a page of help commands by pressing h, scroll forward a page by pressing space, or go forward or backwards one line at a time by pressing j or k. To exit more or less, press q (for quit). If you followed the section on Gene Ontology, you know that the GO files have a relatively simple format to them. . . . . . . . . . . However, the official GRCh37 comes with a mitochondrial sequence 2bp longer than rCRS. . . They often run open-source tools via a GUI. . . 34.2 What is a standout feature of the g:Profiler? . . . . . . . . . 735 XXVI METAGENOMICS 738 114Introduction to metagenomics 114.1What is metagenomics? To some extent yes. . . 10.8 How much Unix do I need to know to be able to progress? . . . . . . . . . SnpSift filter command below selects sites where 3 or more samples have heterozygous genotypes. But what if the object was not accessible - we would have to infer its weight from observing, other related information. These can be standalone software that runs on your local system - they are compatible with web-based applications. Unix is particularly suited to working with such files and has several powerful (and flexible) commands that can process your data for you. Reading this book will teach you what bioinformatics is all about. 27.4 What will our data tell us? . . . Thus to perform the analysis, you already have two scripts: zika-reference.hs4 to download the references zika-data.hs5 to download the sequencing data from SRA We reuse the variable naming of that script and continue with the alignment phases. . . . . . . . . . See the section on p-values later on this page. . . . . $(".owl-carousel").owlCarousel({ . . The Biostar Handbook introduces readers to bioinformatics. . . . . . . . . Most formats may appear to be good enough when first proposed, yet the fast advancements may render them quickly inadequate. . . . 70.4. 449 449 450 450 451 452 . . ACCESS YOUR ACCOUNT 35 . . . . . . 76.3 What is the CRAM format? . We recommend the original resource as an alternative tutorial and source of information. . . When under the pressure to deliver results, and, when faced with the complexity and messiness of real biological data, statistics was, is and will be misused and misinterpreted even by very guardians and the experts that later chide us for not using the concepts in the proper context. . WORKING WITH BAM FILES 14480 as proper pairs whereas bamtools stats reports 15216 for the same quantity. . Is there a list of QC tools? . . . . . . . . . . . . . . . . . 6.2 How much computing power do we need? . . . Of all errors that you may make, this one, and its variations: what is divided by what? . . When this probability is low (small) we can reject the null hypothesis, and we conclude that there is a change. . . The best way to identify a sequence as an enhancer is to experimentally disrupt it and observe gene expression changes. . . . . Almost always we want to answer the question of whether a genes expression level has changed. . No, some files wont be visible via the Finder. . These 3 lines are then sent to the wc command as before. . WILL I NEED TO ACCESS THE SO DATA DIRECTLY? . . . . . . . . The gene association file for human gene products can be obtained as: # Get the file. . . . . . CLASSIFICATION BASED RNA-SEQ OF CONTROL SAMPLES Alignment-based methods word on both genome or transcriptome. . Where are you and what have you got? . . . . . . . . . . . . . . . . . 65.9 How do I choose the scores? . . . HOW DOES READ QUALITY TRIMMING WORK? . . . The answers to this question are always a bit complicated and depend on the goals of the experiment. E.g. . . BLAST USE CASES # Run the blast database builder. 55 55 55 56 56 56 57 58 58 59 59 60 6 How is bioinformatics practiced? . . . What goes into a high-throughput sequencing experiment may be familiar to many wet-lab scientists. . . . For all their visual flair and seeming logic sequence logos can often be misleading. Modern bioinformatics uses mainly the following languages: C - Compiled. Sometimes, you may get sorted bam files, which can be used directly to call peaks. . . . . . . . . 31.7 What is a Functional Class Scoring (FCS)? For example, one common correction is to shorten each transcript end by half of the read length. . . . . . . . . . . . . . . . . . . We believe that even the scientific language is dynamic and we should accept the reality that most scientists use the word SNP to refer to polymorphisms where the frequency in a population is not known. VCF files: samtools.vcf freebayes.vcf gatk.vcf It ran three variant callers and created quite a few files, but the ones we are interested in are: 1. samtools.vcf 1 http://data.biostarhandbook.com/variant/compare-variant-callers.sh 93.4. . blastdbcmd -db ~/refs/refseq/16SMicrobial -entry 'NR_118889.1' -range 1-20 -outfmt "%s" This produces the following output: GGTCTNATACCGGATATAAC 70.7 How do I process all entries in a database? 791 XXVII Appendix 792 121.4Do I need to compute and discuss p-values? . . . . 399 59 Programming with Awk 59.1 Why is Awk popular with bioinformaticians? . . . . . . 47.15Where can I download Illumina software? . . The most well-known is called MACS: Model-based Analysis for ChIP-Seq. . . . . Conceptually, the format is very similar to FASTA but suffers from even more flaws than the FASTA format. In other cases, a more fine-grained analysis will be necessary wherein abundances are quantified for each transcript level. . . . . . . . The skill of using Unix is not just that of understanding the commands themselves. . . . . . . . The idea behind the Phred score is to map two digit numbers that represent error likelihoods to single characters so that the length of the quality string stays the same as the length of the sequence. . . . . . . . . . . . . 55.3 What is the primary concern with duplicates? For example the subunit B of the Cholera Toxin protein complex responsible for the massive, watery diarrhea characteristic of cholera infection has the following sequence: MIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAI ITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAI SMAN It starts with an M, the Methionine, which as we just stated is also the start codon. efetch -db=nuccore -format=fasta -id=AF086833 > AF086833.fa # efetch can take additional parameters and select a section of the sequence. . . You will have a hard time tracking down these errors unless you can visualize the two normally invisible characters. . These files typically represent the results of aligning a FASTQ file to a reference FASTA file and describe the individual, pairwise alignments that were found. . . . Following protocols by the letter is usually entirely counterproductive. . The d at the start of each line indicates that these are directories. . . . 130 13.7 What is a tarbomb? . What are problems with the ORA analysis? 326 Figure 45.3 CHAPTER 45. Instead, we are going to use another nice tool deeptools27 for this task. Other GUI terminal emulators may do the same, but most tend not to. 125.5 Whats the best setup for multiple shell profiles? . . . . . Newer versions of bcl2fastq do not put the Barcode in the file name by default, so it may not always be present. . 99 99 101 101 102 102 103 103 104 105 105 106 107 107 108 108 109 109 110 110 111 111 112 112 113 113 114 115 116 116 . . . . 91.9 How do I extract per-sample information from a multi-sample VCF file? . . Each of the variant callers produces a DIFFERENT variant call. . You may stop for a second and think about whether this ORF naming convention is an optimal long term solution (hint: it is not). . . . . . Phenotypes where the molecular origin is known: cat mimTitles.txt | grep 'Number' | wc -l # 4869 Phenotypes where the molecular origin is unknown: cat mimTitles.txt | grep 'Percent' | wc -l # 1615 What are the phenotypes of unknown molecular origin: cat mimTitles.txt | grep 'Percent' | head Producing the file: Percent Percent Percent Percent Percent Percent Percent Percent Percent Percent 100070 100600 100820 101850 102100 102150 102300 102350 102510 102800 AORTIC ANEURYSM, FAMILIAL ABDOMINAL, 1; AAA1 ANEURYSM, ABDOMINAL AORTIC; A ACANTHOSIS NIGRICANS ACHOO SYNDROME AUTOSOMAL DOMINANT COMPELLING HELIOOPHTHALMIC OUTBURST SYNDR PALMOPLANTAR KERATODERMA, PUNCTATE TYPE III; PPKP3 ACROKERATOELASTOIDOSI ACROMEGALOID CHANGES, CUTIS VERTICIS GYRATA, AND CORNEAL LEUKOMA ROSENT ACROMEGALOID FACIAL APPEARANCE SYNDROME AFA SYNDROME;; THICK LIPS AND ORAL MU RESTLESS LEGS SYNDROME, SUSCEPTIBILITY TO, 1; RLS1 ACROMELALGIA, HEREDITA ACROMIAL DIMPLES SUPRASPINOUS FOSSAE, CONGENITAL ACROPECTOROVERTEBRAL DYSPLASIA; ACRPV F SYNDROME ADENOSINE TRIPHOSPHATASE DEFICIENCY, ANEMIA DUE TO The file can be searched (case insensitive) for diseases: cat mimTitles.txt | egrep -i sickle to produce: NULL 143020 HPA I RECOGNITION POLYMORPHISM, BETA-GLOBIN-RELATED; HPA1 Number Sign 603903 SICKLE CELL ANEMIA RESTRICTION FRA 83.6. . The download script contains simple downloads from various URLs, then it unpacks the data that was downloaded. These are called hard to sequence regions, but it is not always clear why they are hard to sequence. 3. . . . . . . . . Dr. Albert is currently a Research Professor of Bioinformatics8 at Pennsylvania State University and the Director of the Bioinformatics Consulting Center at this institute. . . . 522 522 524 525 525 525 526 526 XIX SAM reference sheet What are the required SAM columns? . Say what? . . . DO I NEED TO COMPUTE AND DISCUSS P-VALUES? . . . Awk automatically splits the input (see the caveats of default splitting later) into variables named $1, $2, $3 etc. A VCF file is typically created from a BAM file, whereas the BAM file was created from a FASTQ and a FASTA file. . . . . . . . . . . . . . . . . Lets put a file in our home directory (specified by ~, remember) and copy it to the lecture directory (~/edu/lecture): $ touch ~/edu/file3 $ cp ~/edu/file3 ~/edu/lecture/ In Unix, the current directory can be represented by a . . . BLAST DATABASES ftp://ftp.ncbi.nlm.nih.gov/blast/db/ 70.3 Can we automate the download of BLAST databases? . . 33 Using the AGRIGO server 33.1 Authors note . . . . . 19.1 How do I use efetch? . . Now the process of biological interpretation starts. . . . 24.4 189 How to I get the percentage for custom bases? . WHY DID MY MULTIQC FAIL WITH AN ERROR? . Here is a tale of caution when it comes to bioinformatics tools. If we run the ls command we should see something like: ls prints (this depends on what computer you use): Applications Desktop Documents Downloads There are four things that you should note here: 1. 14.5 What is the state of data in bioinformatics? By default, the Finder will now show the home directory that your Terminal opens to. . 94.15 What are the steps of an RNA-Seq analysis? 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. . . 37.8 What is the expression data? That belief is rooted in wishful thinking. 2. 3. When we perform a Google query, we are looking to find documents that contain the words that we are seeking. . . The technology has essentially no size limitations if you manage to keep a molecule intact during the library prep, reads up to hundreds of kilobases can be obtained. . . . Once we have a project accession number, say PRJNA257197, we can use it to search for the data that comes with it. . . . . . . . 35.15What does the Gene ID Conversion Tool do? . . Chapter 102 Classification based RNA-Seq of control samples Pseaudoalignments are a concept introduced to the world by the work of Rob Patro and Carl Kingsford at the Lane Center for Computational Biology at Carnegie Mellon University. . CONTENTS 5 . . . . You would need to create bgzip files yourself when compressing VCF and TABIX files. . Note that we also capture the ID (([ ]+ )) so we can restore it in replacement with the capture variable ${1} (more robust than $1). . . . . 92.4 What kind of variant annotators can I use? . . . 8.11 How do I verify that all other programs work? . . . The expression reject the null hypothesis may seem like mincing words or trying to sound clever. . Well, it means get the entire process moving - not just the beginning - the whole thing.