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 Kraken classifier work?......Page 776
How do I match back the bacteria?......Page 778
How to use sourmash?......Page 779
How do I assemble metagenomes?......Page 781
How do I get more information on the project?......Page 782
How many species were present?......Page 784
Does quality control improve the classification?......Page 785
How do I use sourmash to understand my data?......Page 786
How to classify all the data for the project PRJNA46337?......Page 788
What is the most common misunderstanding (paradox) of metagenomics?......Page 790
How reliable are these results?......Page 791
XXVII Appendix......Page 792
Do I need to compute and discuss p-values?......Page 794
What other App Store software is needed?......Page 795
What is Homebrew?......Page 796
How do I upgrade software?......Page 797
How do I view my home directory in the Finder?......Page 798
What is the next step?......Page 800
What are the required libraries for Linux?......Page 801
What is the next step?......Page 802
How do I start Ubuntu Linux?......Page 803
What does not work on Windows?......Page 804
How do I finish setting up Ubuntu on Windows?......Page 805
What are shell profiles?......Page 806
What's the best setup for multiple shell profiles?......Page 807
What should my .bashrc file contain?......Page 808
How do I activate a shell profile?......Page 809
Troubleshooting the PATH......Page 810
Solution 1: Use the full program path......Page 812
Solution 3: Create shortcuts......Page 813
Testing......Page 815
Source code installation......Page 816