A Alternative splicing computational analysis, 114 data processing, 106 experimental design, 114 isoform quantification AltAnalyze, 109 CuffDiff, 110 DEXSeq, 108 DiffSplice, 109 exon/transcript isoform, 108 MATS, 109 MISO, 110 SplicePlot, 110 111 SpliceR, 109 SplicingCompass, 109 read assembly, 107 108 read mapping, 106 107 software recommendations, 115 visualising tools, 111 wet-lab procedure workflow, 113 B Bioanalyzer electropherogram, 304 Bioinformatics metatranscriptomics (see Metatranscriptomics ) nanocage data base calling, 79 data quality, 77 data uploading, 85 EdgeR, 85, 88 5 end position, 82 gene and sample clustering, 82 84 gene expression, 81 82, 84, 89 honey bee samples, 83, 85 Illumina HiSeq sequencing, 77 normalization method, 81 post-mapping filtering, 80 quality and adapter trimming, 79 80 quality control, 79 read count matrix, 80 81 sequence alignment tools, 80 sequencing depth, 79 statistics, 85, 88 3 end position, 82 tools, 85 87 Tophat, 85 workflow, 77, 78 RNA-seq full-length cdna, 93, 95 honey bee genome, 96 IGV genome browser, 96 normalization technique, 92 splice-aware alignment tools, 92 splicing analysis, 93 target enrichment computational resource requirement, 59 60 phylogenomics, 57 59 population genomic analyses, 59 pre-sequencing, 56 57 BluePippin software, 301 Springer International Publishing Switzerland 2016 A.M. Aransay, J.L. Lavín Trueba (eds.), Field Guidelines for Genetic Experimental Designs in High-Throughput Sequencing, DOI 10.1007/978-3-319-31350-4 393
394 Index C Cap-Trapping method, 76 Cell isolation methods FACS, 345 LCM, 345 microfluidic devices, 346 micromanipulation, 344 Chromatin immunoprecipitation followed by sequencing (ChIP-seq) antibody specificity, 229 controls distinct controls, 233 experimental, 231 input control, 232, 233 technical, 232 definition, 224 differential binding analysis occupancy analysis, 247 249 quantitative analysis, 249 250 RNA transcription, 247 downstream analysis, 251 252 ENCODE project, 223, 224 experimental design, 233, 234 functional genomics, 226 occupancy mapping vs. quantitative affinity, 227 229 peak calling annotation, 244 competing methods, 243 definition, 242 deriving consensus peaksets, 244 identification, 243 MACS peak caller, 243 quality assessment, 245 247 tamoxifen resistance, 243 windowing schemes, 245 processing, 225, 226 punctate vs. broad enrichment, 227 read depth, 236 read length, 236 read processing, 240 alignment, 237 blacklists and greylists, 239 duplication, 238 239 FASTQ, 237 FastQC, 237 mapping quality, 238 quality assessment (see Quality assessment ) replication biological replication, 230 experimental replication, 230 high-quality replicates, 231 in vitro experiments, 231 in vivo experiments, 231 RNA-seq, 229 technical replication, 230 samples preparation, 234 235 single vs. paired end, 235 Coding DNA (cdna), 3 Coding region (CDS), 178 Copy number variants (CNVs), 29, 375 Covaris S-series system, 299 Cycloheximide, 183 D Diethyl pyrocarbonate (DEPC), 320 DiffBind package, 240, 250 Differentially expressed genes (DEGs), 84 DNA methylation base-pair and regional level, 213, 214 computational analysis adapter sequencing, 207 assay-specific issues, 208 base qualities, 206 bisulfite sequencing, 205, 206 conversion rate, 208 coverage, 207 regions, 210 213 segmentation, 208 210 SNPs, 207 genome regulation, 197 198 quantitative analysis development, 199 Infinium 450K BeadChip, 199 MeDIP/MBD-seq, 199 202 RRBS, 203 Sanger sequencing, 199 target-bs, 204 205 WGBS, 202 203 DNA-protein interactions. See Chromatin immunoprecipitation followed by sequencing (ChIP-seq) DNA quantification equipment, 296 Nextera protocol, 297 Qubit Fluorometer, 296 DNA sequencing, 2 3 Downstream analysis, 251 252 E Electrophoretic Mobility Shift Assay (EMSA), 262 ENCODE project, 223, 224 European Nucleotide Archive (ENA), 34
395 F FASTQ format, 16 FastX Toolkit, 186, 207 Fisher s exact test, 84, 210 Fluorescence-activated cell sorting (FACS), 345 G Gene regulation, 5 Genome resequencing method, 3 Genome-wide analysis. See DNA methylation GreyListChIP, 239 H Hidden Markov models (HMM), 209 I Illumina platforms, 186 Illumina sequencing, 71 Individual-nucleotide resolution CLIP (iclip), 270 L Laser-capture microdissection (LCM), 345 Logistic regression, 212 Long noncoding RNAs (lncrnas) capture-seq, 161 computational analysis, 165 computational characterization ChIP-seq and DNaseI hypersensitivity, 159 circular RNAs, 160 RNA editing, 159 experimental design, 166 in situ RNA-Seq, 163 isoforms, 156 MET gene, 161 ORF analysis, 156 157 post-analysis validation, 160 protein domain databases, 157 158 ribosome profiling, 158 RNA-seq data sharing, 156 design, 145 differential expression analysis, 153 155 illumina technology, 148 149 mapping/alignment, 150 preprocessing step, 149 protein-coding genes, 153 quality control, 149, 155 read mapping, 152 153 ribosomal RNAs, 153 sequencing libraries, 146 148 single-cell RNA-seq, 162 163 subcellular localization, 146 transcript level, 155 transcriptome, 150 152 software recommendations, 167 wet-lab procedure, 164 M Magnetic bead-based methods, 185 Metagenomics closed-reference annotation, 306 definition, 291 experimental design, 311 human factor, 308 Nextera library, 295 open-reference annotation, 307 reporting, 308 309 requirements, 295 sample replication, 292 293 sequence complexity, 304 306 sequencing options, 293 294 shotgun sequencing, 292 software recommendations, 311 wetlab protocol molarity and library size, 304 Nextera library, 303 304 positive and negative controls, 296 quality, 304 quantification (see DNA quantification ) storage, 295 TruSeq library (see TruSeq library ) workflow analysis, 307, 308 Meta-sequencing, 5 6 Metatranscriptomics amplicon sequencing, 313 bioinformatic analysis annotation, 329 330 de novo assembly, 328 Mac OSX google, 327 process, 325 quality control, 327, 328 SRA, 332 statistical analysis, 330 331 development, 314 experimental design, 315 317 expressed sequence tags, 314 Illumina platform, 318 NGS, 314 reference metagenome, 315
396 Index Metatranscriptomics (cont. ) RNA-seq experiments, 315 rrna, 314 sequencing depth, 318 319 software recommendations, 336 339 wet-lab cell wall lysis, 321 DNAse I treatment, 321 mrna enrichment, 322 precautions, 319 preparation, 325 RNA purification, 320 RNA quality determination, 322 RNase cleaning, 320 sampling, 320 stabilization, 321 WGS, 313 MicroRNAs (mirnas) experimental design, 136 137 non-genome organism, 128 132 online repository, 127 software recommendations, 138 structural features, 123 125 translation and degradation, 122 MinION charged protein nanopores, 9 implementation, 10 mobile sequencer, 10 USB pendrive, 10 Mixture of isoform (MISO), 110 Motif analysis, 251 Multivariate analysis of transcript splicing (MATS), 109 N National Center for Biotechnology Information (NCBI), 7 Nextera library barcodes, 303 cleanup, 303 Illumina Nextera DNA kit, 303 PCR amplification, 304 tagmentation, 303 Next-generation sequencing (NGS) technology, 15, 125 127 Noncoding RNAs, 4 Non-model organism, 3 O Oxford Nanopore Technologies (ONT), 9, 10 P PARalyzer, 277 Photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) antibody, 275 cdna library preparation, 276 C. elegans, 274, 275 computational analysis, 276 278 follow-up experiments, 278 279 HEK293 cells, 274 in vitro techniques EMSA, 262, 266 SELEX, 266 267 in vivo techniques cdna, 270 fl uorescence, 267 268 iclip, 270 RIP-Chip, 268 269 RNase protection, 275 RNP protein component, 269 thioribonucleoside-labeled RNA, 272 transcriptome-wide scale, 272 UV-crosslinking methods, 269 variantion, 270 photoreactive nucleosides, 274 PTGR process, 261 RBPs, 262 RNA molecules, 275 RNase digestion, 275 scale of, 272 SDS-PAGE, 276 4-thiouridine, 274 Post-transcriptional gene regulation (PTGR), 261 Pre-HTS era, 7 9 PubMed repository, 1 Q Quality assessment annotation and genomic distribution, 242 blacklists, 240 ChIPQC package, 240 fragment length, 241 242 peak calling clustering, 246 247 profiles, 245 reads in, 245 SSD, 240 241 Quantitative analysis Bioconductor package, 249 250 consensus peakset, 249
397 DNA methylation Infinium 450K BeadChip, 199 MeDIP/MBD-seq, 199 202 RRBS, 203 Sanger sequencing, 198 target-bs, 204 205 WGBS, 202 203 enrichment, 249 Venn diagram, 250 Qubit Fluorometer, 296 R Reduced representation bisulfite sequencing (RRBS), 203 Restriction-site-associated DNA makers (RADseq), 47 48 Ribosome profiling bioinformatic analysis, 182 computational analysis alignment, 186 187 biases, 187 functional analysis, 187 188 cycloheximide, 177 databases, 188 datasets, 175 177 definition, 175 5 and 3 -UTRs, 178 sequencing depths, 182 speed, 181 technical and biological replicates, 182 translated region identification, 178 180 translational efficiency, 180 181 wet lab protocol barcoding, 185 cell lysis, 183 linker ligation, 185 nuclease footprinting, 183 RNA fragments, 184 185 rrna depletion, 185 sequencing, 186 TISs, 183 RNA binding domains (RBDs), 261, 262 RNA immunoprecipitation followed by microarray analysis (RIP-Chip), 268 269 RNA-protein interactions, 4 5 RNA-seq. See Transcriptome sequencing (RNA-seq) RNA-sequencing method, 3 4 S Sanger sequencing, 18 Sequence read archive (SRA) BioProject, 386, 387 BioSample, 386, 387 Bulk Center Submission, 386 Experiment, 387 experiment and run, 385 formatting practices, 386 new submission, 387 391 running, 387 submission process, 385 Single-cell DNA sequencing applications, 367 368 cell isolation method, 368 computational analysis, 378 data analysis CNV, 375 data reporting, 375 376 identification, 372 programs, 372 SNV, 373 374 tertiary analysis, 376 experimental design, 379 library construction, 370 372 software recommendations, 380 381 wet-lab procedure, 377 WGA, 369 370 Single-cell genome, 6 Single-cell mrna sequencing broad genetic diversity, 343 bulk methods, 344 cell isolation methods FACS, 345 LCM, 345 microfluidic devices, 346 micromanipulation, 344 cell lysis, 346 347 eukaryotic transcription, 352 HiSeq, 352 initial processing early screening, 353 quantitative units, 355 reference alignment, 353 reporting, 355 tools, 353 transcript models, 354 library preparation barcodes, 350 construction, 349, 350 end-tagging methods, 351 identifiers, 351 in vitro transcription, 349
398 Index Single-cell mrna sequencing ( cont. ) IVT amplification, 349 PCR, 348 template switch, 348 UMIs, 351 whole transcript vs. end-tag, 348 quantitative analysis assessing reproducibility, 356 downstream, 356 358 sensitivity, 355 SCRB-Seq, 353 whole transcript methods, 352 Single-nucleotide variants (SNVs), 373 374 Sortmerna package, 149 Standardized Standard Deviation (SSD), 240 State-of-the-art technology, 9 10 Systematic Evolution of Ligands by EXponential Enrichment (SELEX), 266 T Targeted bisulfite sequencing (Target-BS), 204 205 Target enrichment accuracy, 43 advantages, 43, 44 bioinformatics computational resource requirement, 59 60 phylogenomics, 57 59 population genomic analyses, 59 pre-sequencing, 56 57 cost of baits, 48 cost reduction, 53 55 disadvantages, 43, 44, 49 experimental design genomic regions, 52 hybrids/taxonomic designation, 49 phylogenetic analysis, 49 51 population genomic inferences, 50 targeted loci, 50, 52 UCEs, 52 library preparation, 48 on-target contigs, 60 PCR, 47 RADseq, 47 48 (see Re-sequencing method ) RNA-seq, 45 47 WGRS, 45 workflows, 55 56 Transcriptome profiling, 77 bioinformatics (see Bioinformatics ) CAGE, 70 72, 76 77 experimental design, 72 75 full-length cdna, 73 76 RNA-Seq method, 70, 72, 89 90 sequencing platforms, 71 72 Transcriptome sequencing (RNA-seq), 45 47 data sharing, 156 design, 145 differential expression analysis, 153 155 full-length cdna, 93, 95 honey bee genome, 96 IGV genome browser, 96 illumina technology, 148 149 mapping/alignment, 150 normalization technique, 92 preprocessing step, 149 protein-coding genes, 153 quality control, 149, 155 read mapping, 152 153 ribosomal RNAs, 153 sequencing libraries, 146 148 single-cell RNA-seq, 162 163 splice-aware alignment tools, 92 splicing analysis, 93 subcellular localization, 146 transcript level, 155 transcriptome, 150 152 Translating ribosome affinity purification (TRAP), 184 Translational efficiency (TE), 180 181 Translation initiation sites (TIS), 179 TruSeq library A-tails and adapters, 300 barcodes and multiplexing, 300 insert size determination, 298 299 PCR and size selection, 300 302 U Ultraconserved elements (UCEs), 52 Unique molecular identifiers (UMIs), 351 uracil phosphoribosyltransferases (UPRT), 274 W Wafergens Apollo 324 system, 300 Ward s method, 82 Wet lab protocol barcoding, 185 cell lysis, 183 linker ligation, 185
399 metagenomic (see Metagenomic ) metatranscriptomics (see Metatranscriptomics ) nuclease footprinting, 183 RNA fragments, 184 185 rrna depletion, 185 sequencing, 186 TISs, 183 Whole genome re-sequencing (WGRS), 45 Whole-genome bisulfite sequencing (WGBS), 202 203 Whole-genome sequencing bioinformatics assembly metrics, 33 34 and data processing, 16 de novo genome sequencing, 30 32 empirical per-base coverage, 30 ENA, 34 error correction, 25 experiments/sample collection, 23 genome assembly, 26 28 quality clipping and filtering, 24 25 read mapping, 28 29 redundancy, 30 resequencing, 31, 32, 34 variant calling, 29 bioinformatics and data processing, 16 de novo genome sequencing contigs and scaffolds, 17 diploid organism, 17 heterozygosity, 18 k-mers, 18 quality of, 16 size and complexity, 17 DNA extraction, 14 experimental sequencing, 22 23 genome complexities and sequencing strategy, 20 22 library preparation phase, 15 NGS, 15 PCR-dependent GC bias, 19 recommendations, 14, 17 resequencing, 16 18 sample collection, 14 Whole genome shotgun (WGS), 313 Z ZymoTM Purification Kit, 303