The Biostar Handbook: 2nd Edition

I Preface......Page 32 Online courses......Page 34 Access your account......Page 35 Who is a Biostar?......Page 36 About the author......Page 38 What is this book about?......Page 41 What is covered in the book?......Page 42 What subfields of bioinformatics exist?......Page 43 Is there a list of functional assays used in bioinformatics?......Page 45 But what is bioinformatics, really?......Page 46 Is creativity required to succeed?......Page 47 What type of computer is required?......Page 48 Can I learn bioinformatics from this book?......Page 49 How long will it take me to learn bioinformatics?......Page 50 Biology for bioinformaticians......Page 51 What is DNA?......Page 52 What is sense/antisense?......Page 54 What is a genome's purpose?......Page 55 How does a genome function?......Page 56 What is a protein?......Page 57 What is an ORF?......Page 58 Do genomes have other features?......Page 59 What is homology?......Page 60 What is the recommended computer for bioinformatics?......Page 62 What about the cloud?......Page 63 Are there alternatives to using Unix?......Page 64 What is Bioconductor?......Page 65 What is Galaxy?......Page 66 Are commercial bioinformatics software packages expensive?......Page 67 Should I freelance as a bioinformatician?......Page 68 What do bioinformaticians look like?......Page 69 How do I perform a holistic analysis?......Page 70 What are the rules of a bioinformatics analysis?......Page 71 What does simple mean?......Page 72 How to deal with anxiety and stress?......Page 73 II Installation......Page 75 Is this going to be difficult?......Page 77 What are environments?......Page 78 What is bioconda?......Page 79 How do I check that Entrez Direct works?......Page 80 How do I verify that all other programs work?......Page 81 How do I report installation problems?......Page 82 How do I install a new tool?......Page 83 How should I set up my file structure?......Page 84 What to do if I get stuck?......Page 85 What features should my text editor have?......Page 86 Super annoying behaviors......Page 87 Which text editor to choose?......Page 88 Watch the line endings on Windows!......Page 89 III UNIX COMMAND LINE......Page 90 What does the command line look like?......Page 92 Is the command line hard to learn?......Page 93 What is a shell?......Page 94 What is the best way to learn Unix?......Page 96 Where can I learn more about the shell?......Page 97 Why Unix?......Page 98 1. The Terminal......Page 99 3: The Unix tree......Page 101 5: Making new directories......Page 102 7: The root directory......Page 103 8: Navigating upwards in the Unix filesystem......Page 104 10: Finding your way back home......Page 105 11: Making the ls command more useful......Page 106 13: Removing directories......Page 107 15: Creating empty files with the touch command......Page 108 17: Renaming files......Page 109 19: Removing files......Page 110 21: Copying directories......Page 111 23: Viewing files with cat......Page 112 25: Editing small text files with nano......Page 113 26: The $PATH environment variable......Page 114 27: Matching lines in files with grep......Page 115 Miscellaneous Unix power commands......Page 116 What directory should I use?......Page 118 Where are we getting the data from?......Page 119 How do I obtain a data file that is online?......Page 120 How many feature types are in this data?......Page 125 One-liners......Page 126 What objects may be compressed?......Page 128 How do I compress or uncompress a file?......Page 129 How do I compress or uncompress multiple files?......Page 130 What is a tarbomb?......Page 131 How do we use tar again?......Page 132 IV DATA SOURCES......Page 133 Essential properties of data......Page 135 What is the state of data in bioinformatics?......Page 136 What kind of problems does bioinformatics data have?......Page 137 How complete is the data that will I obtain?......Page 139 Final thoughts on data......Page 140 What are the major DNA data repositories?......Page 142 What kind of other data sources are there?......Page 144 What's in a name?......Page 145 Project systematic names......Page 146 A Quick look at the GENBANK format.......Page 147 A quick look at the FASTQ format......Page 148 A quick look at the GFF/GTF/BED formats......Page 149 Can I convert between formats.......Page 150 What are genomic builds?......Page 151 Should download data first or get data on-demand?......Page 152 How many genomic builds does the human genome have?......Page 153 How do we transfer genomic coordinates between builds?......Page 154 Human gene naming......Page 155 Is there a better resource for human annotations?......Page 156 What can I get from ENSEMBL?......Page 157 What a working strategy for finding reference information......Page 158 What is Entrez?......Page 159 How is data organized in NCBI?......Page 160 How do I use Entrez Direct?......Page 161 How do we search with Entrez Direct?......Page 162 How to do more work with Entrez Direct?......Page 163 How do I use efetch?......Page 164 How do I get run information on a project?......Page 165 How do I extract taxonomy information?......Page 166 V DATA FORMATS......Page 168 Should I re-format (transform) my data?......Page 170 When to re-format (transform) data?......Page 172 What is the GenBank format?......Page 173 How are RefSeq sequences named?......Page 174 What is the FASTA format?......Page 178 Are there problems with this format?......Page 179 Is there more information in the FASTA sequences?......Page 180 Where do I get a fasta file?......Page 181 What is the FASTQ format?......Page 182 How to recognize FASTQ qualities by eye......Page 183 Is there more information in FASTQ headers?......Page 184 How do I convert FASTQ quality codes at the command line?......Page 185 Closing thoughts on FASTQ......Page 186 Advanced FASTQ processing......Page 187 How do I get the GC content?......Page 188 How do I find FASTA/Q sequences containing degenerate bases and locate them?......Page 189 How do I locate motif/subsequence/enzyme digest sites in FASTA/Q sequence?......Page 190 How do I split FASTA sequences according to information in the header?......Page 191 How do I search and replace within a FASTA header using character strings from a text file?......Page 192 How do I extract paired reads from two paired-end reads files?......Page 193 How to concatenate two FASTA sequences in to one?......Page 194 VI VISUALIZING DATA......Page 196 What are the challenges of visualization?......Page 198 Why are default browser screens so complicated?......Page 199 How do I interpret glyphs?......Page 200 What about online genome browsers?......Page 201 What is IGV?......Page 202 What data does IGV come with?......Page 203 How do I create a custom genome in IGV?......Page 204 VII SEQUENCE ONTOLOGY......Page 206 Why is the ontology necessary?......Page 208 Who names the genes?......Page 209 What will our data tell us?......Page 210 Where do I see Sequence Ontology (SO) terms used?......Page 211 How do I access the Sequence Ontology browser?......Page 212 How are the SO relationships defined?......Page 214 How can I quickly search the Sequence Ontology?......Page 215 How to search for other information?......Page 216 VIII GENE ONTOLOGY......Page 218 How is the GO designed?......Page 220 What kind of properties do annotated gene products have ?......Page 221 How are GO terms organized?......Page 222 Where can the visualize GO terms online?......Page 224 What does a GO term file contain?......Page 227 Where can I find the association files for different organisms?......Page 228 What format does the GO association file have?......Page 229 What kind of properties does the GO data have?......Page 230 What are the most annotated human genes and proteins?......Page 231 What are the ten most highly annotated genes in the GO dataset?......Page 232 How complete is the GO?......Page 233 The sorry state of data categorization......Page 235 What is an Over-Representation Analysis (ORA)?......Page 236 Are there different ways to compute ORA analyses?......Page 237 What is a Functional Class Scoring (FCS)?......Page 239 Should I trust the results of functional analyses?......Page 240 What tools are used to perform enrichment analysis?......Page 241 Will different tools produce different results?......Page 242 How do I perform a gene set enrichment analysis?......Page 243 How to use AgriGO......Page 245 How to use SEA?......Page 246 How do I prepare a custom annotation for AgriGO?......Page 247 What is a standout feature of the g:Profiler?......Page 251 What functionality does the g:Profile have?......Page 252 How to use g:profiler at the command line......Page 254 Authors note......Page 256 What are the different steps in a DAVID analysis?......Page 257 How do I start an analysis with DAVID?......Page 258 What is the Functional Annotation Summary?......Page 259 What is a Functional Annotation Chart ?......Page 260 What is Functional Annotation Clustering?......Page 261 What is the Gene Functional Classification Tool?......Page 262 What is an EASE Score?......Page 264 What are some pros and cons of DAVID?......Page 265 Plot GO terms......Page 267 Finding enrichment with goatools......Page 272 How to use ErmineJ......Page 276 What are the annotation files?......Page 277 What is a gene score file?......Page 278 How do I start an analysis in ErmineJ?......Page 279 What is an Over-Representation Analysis (ORA)?......Page 280 What are the results of an ORA analysis?......Page 281 Is multi-functionality good or bad?......Page 283 What is Gene Score Resampling (GSR)?......Page 284 What is Correlation Analysis in ErmineJ?......Page 285 IX REPRODUCIBILITY......Page 286 What is the red herring of reproducibility?......Page 288 Is science really facing a reproducibility crisis?......Page 289 So what does reproducibility mean?......Page 290 What is the best strategy to reproduce results?......Page 291 Are the challenges of data reproducibility recognized?......Page 292 How to get the information for the Ebola paper?......Page 293 Is it possible to access the data for this analysis?......Page 294 Where can we find out more about this dataset?......Page 295 How do we download results for this paper?......Page 296 How to get the information for the Zika paper?......Page 298 How do we find the data for this paper?......Page 299 What chapters cover the Zika data?......Page 301 Will the best bioinformaticians on the planet produce reproducible analyses?......Page 302 What problem does the paper attempt to solve?......Page 303 Where is the data?......Page 304 Is the analysis reproducible?......Page 305 The gods must be crazy......Page 306 Redo: Explore the genome of a paleolithic-era archaic human......Page 308 X SEQUENCING INSTRUMENTS......Page 310 What is in a name?......Page 312 What type of sequencing instruments are in use?......Page 313 How accurate are sequencing instruments?......Page 314 How do sequencing instruments work?......Page 315 Can reads be in different orientations?......Page 316 What is paired-end sequencing?......Page 317 What is mate-pair sequencing?......Page 318 What types of sequencers does Illumina manufacture?......Page 319 What is an SMRT cell?......Page 321 What is the output of a PacBio run?......Page 322 How is the BAM file formatted?......Page 323 What is the per-base quality of PacBio reads?......Page 324 How good are the consensus corrected reads?......Page 325 What is MinKNOW?......Page 327 What is different between 1D and 2D reads?......Page 328 What is HDFView?......Page 329 How can I extract data from a FAST5 file using poretools?......Page 330 A recap of high-throughput sequencing......Page 331 How do I select proper sample identifiers?......Page 332 Why is sample/library QC essential?......Page 333 What does an original Illumina sequence data folder contain?......Page 334 What should I expect to get from a sequencing provider?......Page 335 What should I do with the raw data?......Page 336 Where can I download Illumina software?......Page 337 XI SEQUENCING DATA......Page 338 What are typical coverages for RNA sequencing?......Page 340 How is genome coverage computed?......Page 341 Do theoretical coverages describe reality?......Page 342 What are the SRA naming schemes?......Page 343 How does the sratoolkit work?......Page 344 Where do downloads go?......Page 345 How do we get information on the run?......Page 346 How to automate downloading multiple SRA runs?......Page 347 Is there even more metadata?......Page 349 How do I process the docsum format?......Page 350 How much data in the SRA?......Page 351 How do we extract columns from a comma separated file?......Page 352 How many sequencing runs per organism?......Page 353 How many sequencing runs for each sequencing platform?......Page 354 How many runs per sequencing instrument model?......Page 355 XII QUALITY CONTROL......Page 356 What part of the FASTQ file gets visualized?......Page 358 What is the FastQC tool?......Page 359 Should I be worried about the stoplight symbols?......Page 360 What does the sequence quality visualization tell us?......Page 361 What does the sequence quality histogram tell us?......Page 362 What is the next step after visualizing the quality?......Page 363 What can go wrong with the data?......Page 365 How reliable are QC tools?......Page 366 Is there a list of QC tools?......Page 367 How does read quality trimming work?......Page 368 Can we customize the adapter detection?......Page 370 Why do we need to trim adapters?......Page 371 How do we trim adapters?......Page 372 Do we have to remove adapters from data?......Page 373 How do I cut a different adapter?......Page 374 How do we detect sequence duplication?......Page 375 What does the FastQC duplicate plot mean?......Page 376 How do I remove duplicates?......Page 378 How to combine the results into a single report.......Page 379 Why did my multiqc fail with an error?......Page 380 Can I combine reads into a single sequence?......Page 382 How do I merge reads?......Page 383 How well do mergers work?......Page 385 Is there anything newer than fastqc?......Page 386 How do we correct errors?......Page 387 XIII WRITING SCRIPTS......Page 388 Why should I write scripts?......Page 390 What is refactoring code?......Page 391 Why is it right to stop a script when an error occurs?......Page 392 How do I add runtime parameters?......Page 393 How can I manipulate file paths?......Page 394 How can I build more complicated scripts?......Page 395 What programming languages do Bioinformaticians use?......Page 398 Which programming language should I learn?......Page 399 Why is Awk popular with bioinformaticians?......Page 400 How is the output split into columns?......Page 401 How can I write more complicated Awk programs?......Page 402 Are there any unique Awk patterns I should know about?......Page 403 How can I format the output of Awk?......Page 404 How can I learn more about Awk?......Page 405 What is BioAwk?......Page 406 How to use Awk/BioAwk for processing data?......Page 407 Using bioinformatics recipes......Page 409 Can I run recipes on my computer?......Page 410 How to make best of use of recipes?......Page 411 Can I use the software to run my own recipe website?......Page 412 XIV PATTERNS......Page 413 Can adapters be identified by a pattern?......Page 415 How can I search genomic sequences for patterns?......Page 416 What are regular expressions?......Page 417 What are some challenges I may face when using regular expressions?......Page 418 What are k-mers good for?......Page 420 Should I use the k-mer report produced by FastQC?......Page 421 XV ALIGNMENTS......Page 422 What is a sequence alignment?......Page 424 How are alignments displayed?......Page 425 How are alignments generated?......Page 426 How does alignment scoring work?......Page 427 What kind of scoring matrices are there?......Page 428 What is a CIGAR string?......Page 429 Where can I learn more about alignments?......Page 430 Install the helper scripts......Page 432 What is a global alignment?......Page 433 What is a local alignment?......Page 434 What is a semi-global alignment?......Page 435 Will alignments always indicate the correct variation?......Page 437 XVI BLAST......Page 440 What are the BLAST tools?......Page 442 What are the fundamental concepts of BLAST?......Page 443 How do I use BLAST?......Page 444 What are the blast tools?......Page 446 What is the Blast terminology?......Page 447 What is an E-Value......Page 448 How do I build custom blast databases?......Page 449 How do I format the output differently?......Page 450 What are blast tasks?......Page 451 Will blast find all alignments?......Page 452 Can we download BLAST databases?......Page 455 Can we automate the download of BLAST databases?......Page 456 How do I reformat sequences in a BLAST database?......Page 457 How do I process all entries in a database?......Page 458 XVII SHORT READ ALIGNMENT......Page 460 What are short read aligners?......Page 462 What are the limitations of short read aligners?......Page 463 How do we pick the best short read aligner?......Page 464 What features do we look for in a short read aligner?......Page 465 How does bwa work?......Page 466 How do I use bwa?......Page 467 How do I find help on bwa?......Page 468 How do I build an index with bowtie?......Page 470 How do I align with bowtie?......Page 471 How can I tell which aligner is better?......Page 473 How do I choose the right aligner?......Page 475 How do I align more than two sequences?......Page 476 What programs can be used to align multiple sequences?......Page 478 XVIII SAM/BAM Format......Page 479 What is a BAM file?......Page 481 Is it SAM, BAM or CRAM now?......Page 482 What information is stored in a SAM file?......Page 483 How do I create SAM/BAM files?......Page 484 Can unaligned data be stored in a SAM file?......Page 485 How to make a BAM file......Page 487 Where do I get a BAM file?......Page 491 What is the SAM header?......Page 492 Column 2: FLAG......Page 494 Columns 3-4: Reference Name (RNAME) and Position (POS)......Page 498 Column 5-6: Mapping Quality (MAPQ) and Compact Idiosyncratic Gapped Alignment Representation (CIGAR)......Page 499 Columns 7-9: RNEXT, PNEXT, TLEN......Page 500 Columns 10-11: SEQ and QUAL......Page 501 What do the SAM tags mean?......Page 502 Where do I get a BAM file?......Page 503 How can I extract a section of the BAM file?......Page 504 How do I valid (mapped) alignments?......Page 505 How do I select forward or reverse alignments?......Page 506 How to get an overview of the alignments in a BAM file?......Page 507 What is a proper-pair?......Page 509 How do I use flags?......Page 510 How do I combine multiple flags?......Page 511 How do I filter on mapping quality?......Page 512 How can I find out the depth of coverage?......Page 513 What is a SUPPLEMENTARY alignment?......Page 514 What is a PRIMARY or REPRESENTATIVE alignment?......Page 515 What do the flags mean?......Page 516 What do the SAM header tags mean?......Page 517 What do the SAM alignment tags mean?......Page 519 What are BWA aligner specific tags?......Page 520 How to mutate a sequence from the command line?......Page 522 How do I reconstruct alignment from CIGAR and MD strings?......Page 524 How long is an alignment?......Page 525 How to visualize a SAM file as pairwise alignment?......Page 526 XIX Genomic Variation......Page 527 How do we classify variants?......Page 529 Is the SNP term used consistently?......Page 530 What is a haplotype?......Page 531 Can the OMIM data be obtained?......Page 532 What terms and concepts does OMIM know about?......Page 533 How many phenotypes of Mendelian inheritance?......Page 534 How many unique pairs of gene and phenotypes are there?......Page 535 What genes have the most annotated phenotypes?......Page 537 What types of data simulators exist?......Page 538 How do I simulate sequencing data with dwgsim?......Page 539 How do I mutate a sequence with msbar?......Page 540 How do I simulate mutations with biosed?......Page 541 How do I simulate reads with art?......Page 542 The reference genome is not real......Page 544 What would perfect data look like?......Page 545 What would realistic and useful data look like?......Page 546 What would a deletion from the genome look like?......Page 547 How can you tell when parts of the genome are swapped?......Page 548 What if the genome contains new regions?......Page 549 What if we reverse complement parts of the genome?......Page 550 XX Variation Calling......Page 552 What is the ploidy?......Page 554 What is the best method to call variants?......Page 555 How do I generate VCF files?......Page 556 How do I prepare the reference genome for variant calling?......Page 559 How do I align sequencing data against a reference?......Page 560 How do I use the FreeBayes variant caller?......Page 561 How to install GATK?......Page 562 How do I use GATK?......Page 563 Can I customize the variant calling process?......Page 564 How do I perform multi-sample variant calling process?......Page 565 How do I interpret the visualization?......Page 566 What is variant normalization?......Page 567 How do I normalize a variant?......Page 568 How important is it to understand the VCF format?......Page 569 What do the words mean?......Page 570 What are VCF records?......Page 571 What is represented in the REF/ALT columns.......Page 572 What are genotype likelihoods?......Page 573 What are additional resources?......Page 574 How can I filter VCF files?......Page 575 How do I extract all variants from a particular region?......Page 576 How do I get variants present in all samples?......Page 577 How to print genotype (GT) of all samples at each position.......Page 578 How do I get variants for which allele count is above a specific value?......Page 579 How do I select variant sites based on quality and read depth?......Page 580 How do I find variants unique to one sample but absent in all other samples?......Page 581 Are are consequences of variant effects?......Page 582 What kind of variant annotators can I use?......Page 583 How do I use snpEff?......Page 584 Can I build custom annotations for snpEff?......Page 585 How do I use the Ensemble Variant Effect Predictor (VEP)......Page 586 Why is it so difficult to call variants?......Page 587 How to explore the effect of mutations on variant callers?......Page 588 What happens when the variation is more complicated?......Page 589 XXI RNA-SEQ PRINCIPLES......Page 591 What is RNA-Seq when reduced to its essence?......Page 593 What is RNA-Seq analysis?......Page 594 What are the main methods for quantifyin RNA-Seq reads?......Page 595 What complicates RNA-Seq analysis?......Page 596 What kind of splicing events exist?......Page 597 How many replicates do I need?......Page 598 Will there ever be an optimal RNA-Seq method?......Page 599 What is a splice-aware aligner?......Page 600 Which splice-aware aligner should I use?......Page 601 How do I compare mRNA abundances?......Page 602 What is a library size normalization?......Page 603 What is the RPKM?......Page 604 What the FPKM is that?......Page 605 What is TPM?......Page 606 What is a spike-in control?......Page 607 How should I name samples?......Page 608 Why do statistics play a role in RNA-Seq?......Page 610 What kind of questions can we answer with a statistical test?......Page 611 What is R?......Page 612 What is Bioconductor?......Page 613 What does a p-value mean?......Page 614 So how do I deal with p-values?......Page 616 Do I need to compute and discuss p-values?......Page 617 How can I run RNA-Seq differential expression scripts from the command line?......Page 618 What is the norm-matrix file?......Page 619 How can I plot my normalized matrix?......Page 620 How would I even solve this puzzle?......Page 622 The Prestige......Page 623 How to solve it (a hint)......Page 624 XXII RNA-SEQ EXAMPLE......Page 625 What type of data is included?......Page 627 How do I download the example data?......Page 628 What is a spike-in control?......Page 630 How do I align an RNA-seq sample?......Page 631 How do I automate my code for all samples?......Page 632 How to better automate the process?......Page 633 How do I estimate the abundance for a single sample?......Page 634 Are there different ways to count overlaps?......Page 635 How do I find differential expression?......Page 636 What does a differential expression file look like?......Page 637 Did our RNA-Seq analysis reproduce the expected outcomes?......Page 638 How do I generate the list of differentially expressed features?......Page 640 What does the differential expression file look like?......Page 641 How do I interpret the results?......Page 642 What is the main difference between alignments and classification based RNA-Seq?......Page 643 How do I quantify transcripts with Kallisto?......Page 644 How do I quantify all samples with Kallisto?......Page 645 How do I run a differential expression study?......Page 646 XXIII RNA-SEQ ZIKA......Page 647 What data does the project contain?......Page 649 How to obtain the data?......Page 650 What will the accession number files contain?......Page 652 How do I analyze data generated on different sequencing platforms?......Page 653 How to get the reference genomes?......Page 655 What are the steps to align the data?......Page 656 How do I generate feature counts?......Page 658 How do I visualize the differentially expressed genes?......Page 659 What is the result of the differential expression analysis?......Page 661 How do the results relate to expected results?......Page 662 How do I build a Kallisto index?......Page 664 How do I quantify transcripts with Kallisto?......Page 665 How do I run kallisto for all the samples?......Page 666 How do I quantify the results?......Page 667 How do I find differentially expressed transcripts?......Page 668 XXIV CHIP-SEQ Analysis......Page 670 What are the challenges of ChIP-Seq?......Page 672 How are RNA-Seq studies different from ChIP-Seq studies?......Page 673 What does a peak represent?......Page 674 How do we determine peaks from sequencing data?......Page 676 What does a ChIP-Seq data measure?......Page 677 What type of ChIP-Seq studies are performed?......Page 679 Should my ChIP-Seq aligner be able to detect INDELs?......Page 680 What are the processing steps for ChIP-Seq data?......Page 681 How do I obtain the data for project PRJNA306490?......Page 682 How do I find out more information each sample?......Page 684 How do I get the standard yeast genome data and annotation?......Page 685 Do I need to trim data to borders?......Page 686 How do I visualize the alignments?......Page 687 Are there other ways to generate bigwig files?......Page 689 What is the next step of analyzing the data?......Page 690 How do I reanalyze a ChIP-Seq experiment?......Page 691 Should I first summarize my data ?......Page 692 What tools can I use to predict peaks?......Page 693 What else can I do to refine the peaks?......Page 694 How did the paper call peaks?......Page 695 What makes motifs difficult to evaluate?......Page 696 How do I find known motifs?......Page 697 How do I call motifs?......Page 699 Have motifs ever misled scientists?......Page 701 XXV Ming Tang's ChIP Seq......Page 703 What are the processing steps for ChIP-seq data?......Page 705 Data sets......Page 706 How to obtain the data?......Page 707 How to align ChIP-seq reads?......Page 708 How do I call peaks?......Page 709 How do I call super-enhancers?......Page 710 How do I get a normalized bigWig track for visualizing the raw signal?......Page 711 How do I put all the steps together?......Page 712 What are the black-listed regions?......Page 716 How do I visualize the peaks?......Page 717 How do I compare the peak sets?......Page 718 How do I annotate my peaks?......Page 720 How do I do pathway enrichment analysis for the peaks?......Page 722 How do I do motif analysis with the peaks?......Page 724 How to call differential peaks?......Page 726 How do I generate a heatmap with ChIP-seq data?......Page 728 How do I generate a meta profile plot with ChIP-seq data?......Page 730 Where can I get public available ChIP-seq data sets?......Page 735 XXVI METAGENOMICS......Page 738 What is metagenomics?......Page 740 Do humans host ten times as many bacteria as their cells?......Page 741 What type of answers does metagenomics produce?......Page 742 What is whole-genome metagenomics?......Page 743 How many bacteria are unknown?......Page 744 What online tools can I use?......Page 746 What command line tools may be used to analyze metagenomics data?......Page 747 What are typical steps of an analysis?......Page 748 What the heck is an OTU?......Page 750 What is the story with the NCBI disclaimer?......Page 751 How to get the NCBI taxonomy file?......Page 752 How many taxonomical ranks are there?......Page 753 How do I search the taxonomies?......Page 754 How do I find the taxid of a taxonomy name?......Page 755 How do I get the lineage of a taxid?......Page 756 How many species of viruses are in the current taxonomy?......Page 757 How do I get all known bacterial genomes?......Page 759 Are there other ways to get all bacterial/viral genomes?......Page 760 How do I set up the BLAST for taxonomy operations?......Page 761 What does the env_nt blast database contain?......Page 762 What data will be analyzed?......Page 764 How do I visualize the 16S classification?......Page 765 Are there better ways to classify 16S sequences?......Page 766 Are there other 16S classification methods?......Page 767 How do I classify multiple samples?......Page 768 How do I determine statistically relevant differences between the classification counts?......Page 769 How do I evaluate how well a given method works?......Page 772 How do I obtain the data?......Page 773 What are the expected abundances of the data?......Page 774 What is the coverage?......Page 775 How does Kra