PowerPoint-Präsentation

PowerPoint-Präsentation

V8 Genomics data Program for today: - Read mapping - SNP calling - SNP frequencies in 1000 Genomes data - Isoforms of genes (alternative splicing) - Non-canonical translation - Removing sequence redundancy V8 Processing of Biological Data 1 (1) Read mapping: range of usage The accurate alignment of reads generated by NGS machines to a reference genome is a crucial part in many application workflows, such as - genome resequencing (in contrast to de novo assembly), - DNA methylation, - RNA-Seq (transcriptomics), - ChIP sequencing (e.g. histone marks, TFBS occupancies), - SNP detection, - detection of genomic structural variants, and - metagenomics (sequencing mixtures of organisms). Hatem et al. BMC Bioinformatics (2013) 14:184 V8

Processing of Biological Data 2 Read mapping tools Numerous tools have been developed for this challenging task: MAQ, RMAP, GSNAP, Bowtie, Bowtie2, BWA, SOAP2, Mosaik, FANGS, SHRIMP, BFAST, MapReads, SOCS, PASS, mrFAST, mrsFAST, ZOOM, Slider, SliderII, RazerS, RazerS3, Novoalign and GPU-based tools such as SARUMAN and SOAP3. Hatem et al. BMC Bioinformatics (2013) 14:184 V8 Processing of Biological Data 3 Read mapping techniques: (1) Hash tables For most of the existing tools, the mapping process starts by building an index either for the reference genome or for the reads. Then, the index is used to find the corresponding genomic positions for each read. There are two main types of techniques for this: Hash tables + BWT (1) The hash based methods either hash the reads or the genome. The main idea for both types is to build a hash table for subsequences of the

reads/genome. The key of each entry is a subsequence while the value is a list of positions where the subsequence can be found. Hatem et al. BMC Bioinformatics (2013) 14:184 V8 Key Hashed index Genomic location GCTAGC Key1 Chr1 123412 TTTAGC KeyN Chr6 988472 Processing of Biological Data

4 Read mapping techniques: (2) Burrows Wheeler transform The BWT of the string T = "abracadabra$" is "ard$rcaaaabb. It is represented by the matrix M where each row is a rotation of the text, and the rows have been sorted lexicographically. The transform corresponds to the last column labeled L. Modern alignments use an extension of BWT named FM index after Ferragina & Manzina I 1 2 3 4 5 6 7 8 9 10 11 12 F $

a a a a a b b c d r r abracadabr $abracadab bra$abraca bracadabra cadabra$ab dabra$abra ra$abracad racadabra$ adabra$abr abra$abrac a$abracada acadabra$a L a r d

$ r c a a a a b b www.wikipedia.org V8 Processing of Biological Data 5 Read mapping techniques: (2) Burrows Wheeler transform C[c] is a table that, for each character c in the alphabet, contains the number of occurrences of lexically smaller characters in the text. C[c] of "ard$rcaaaabb" c C[c] $ 0 a 1

b 6 c 8 d 9 r 10 The function Occ(c, k) is the number of occurrences of character c in the prefix L[1..k]. Occ(c, k) of "ard$rcaaaabb" a r d $ r c a

a a a b b 1 2 3 4 5 6 7 8 9

10 11 12 $ 0 0 0 1 1 1 1 1 1 1 1

1 a 1 1 1 1 1 1 2 3 4 5 5 5

b 0 0 0 0 0 0 0 0 0 0 1 2 c 0

0 0 0 0 1 1 1 1 1 1 1 d 0 0

1 1 1 1 1 1 1 1 1 1 r 0 1 1 1

2 2 2 2 2 2 2 2 www.wikipedia.org V8 Processing of Biological Data 6 Read mapping techniques: (2) Burrows Wheeler transform The FM-index itself is a compression of the string L together with C and Occ in some form, as well as information that maps a selection of indices in L to positions in the original string T. FM index is used e.g. by the tools Bowtie and BWA

Soap uses a different variant of BWT. www.wikipedia.org V8 Processing of Biological Data 7 Read alignment: features Crucial default options: Maximum number of mismatches in the seed (default 2). The seed are the first few tens of base pairs of a read. The seed part of a read is expected to contain less erroneous characters. - Maximum number of mismatches in the read (2 to 10) - Seed length (28 32). - Quality threshold: It is equal to 70 for MAQ and Bowtie while it depends on the read length and the genome size for Novoalign. -

Splicing: This option is enabled for GSNAP. - Gapped alignment: It is enabled for Bowtie2, GSNAP, BWA, Novoalign and MAQ while it is disabled for SOAP2. - Minimum and maximum insert sizes for paired-end mapping: The insert size represents the distance between the two ends. (0 to 500) Hatem et al. BMC Bioinformatics (2013) 14:184 V8 Processing of Biological Data 8 Read alignment: evaluation criteria The sequence in blue is the original genomic position where the simulated read was extracted from. After applying sequencing errors, the read does not exactly match to the original location (3 mismatches). 3 possible alignment locations for the read with their mapping quality score (MQ). Nave criterion: only consider the alignment (1) as the correct alignment.

Hatem et al. BMC Bioinformatics (2013) 14:184 V8 Processing of Biological Data 9 Read alignment: evaluation criteria Ruffalo et al. criterion: consider also the mapping quality. If the used threshold is 30, then (1) is correctly mapped while (2) and (3) are incorrectly mapped-strict. If the threshold is 40, then (3) is considered as incorrectly mapped relaxed (no correct mapping available higher than the threshold). Holtgrewe et al. criterion: considers all matches with distance k. Here, it would detect (1) and (2) and consider them correctly mapped while (3) would be considered as incorrectly mapped. Hatem et al: We define a read to be correctly mapped if it is mapped while not violating the mapping criteria. V8 Hatem et al. Processing of Biological Data BMC Bioinformatics (2013) 14:184 10 Read alignment: throughput for simulated data Task: map 1 million reads of length 125 extracted from the Human genome

using wgsim with 0.09% SNP mutation rate, 0.01% indel mutation rate, and 2% uniform sequencing error rate. BWA-ND Each tool was used with its own default options. Bowtie Bowtie only maps 68% of the reads, but achieves high throughput. BWA BWA maps 91% of the reads, but 15 x lower throughput. However, when used with the same options as Bowtie, BWA achieves even a higher performance. V8 BWA-ND refers to BWAs results while using Bowties default options which are 2 mismatches in the seed, 3 mismatches in the whole read, and no gapped alignment. Hatem et al.

Processing of Biological Data BMC Bioinformatics (2013) 14:184 11 Read alignment: number of allowed mismatches Mapping 1 million reads of length 125 extracted using wgsim from the Human genome while allowing up to 7 mismatches and a quality threshold of 140. The error is 0.6% for SOAP2 and MAQ and 0.45% for GSNAP. Hatem et al. BMC Bioinformatics (2013) 14:184 V8 Processing of Biological Data 12 Read alignment: comparison on real data Comparing the different tools while changing the maximum allowed mismatches (Tmms) from 2 to 7. A real mRNA data set of 1 million reads of length 51 bps extracted from the Spretus mouse strain and mapped against the mouse genome version mm9 was used in this experiment. Hatem et al. BMC Bioinformatics (2013) 14:184 V8

Processing of Biological Data 13 Read alignment: effect of read length The effect of changing the read length from 36 to 500. The reads were extracted from the Human genome. Longer reads tend to have more mismatches. For a fixed number of mismatches, the read length decreases the percentage of mapped reads. Hatem et al. BMC Bioinformatics (2013) 14:184 V8 Processing of Biological Data 14 Read alignment: SNP calling with different mappers Tools Log-odds ratio 5 100 200

300 400 500 600 700 800 900 1000 1000000 Bowtie 89479 24337 5082 2231

1076 648 426 281 0 0 0 1171 Bowtie2 200914 62178 10018 4200 2052 1156 767

537 0 0 0 2035 BWA 192050 52115 9028 4049 1894 1087 737 525 0

0 0 2067 SOAP2 174475 49302 8552 3824 1837 1030 704 508 0 0 0 1941

Novoalign 69798 4061 1875 936 519 363 252 0 0 0 941 GSNAP 207920 69015

11416 4928 2482 1325 971 617 0 0 0 2602 17586 The tools were used to map an mRNA dataset of 23 million reads extracted from the Spretus mouse strain. Then Partek is used to detect SNPs against mouse genome version mm9. A quality threshold of 70 was used for Bowtie and Novoalign while the remaining tools were allowed 5 mismatches. Each column shows the number of SNPs detected with a log-odds ratio, which is a measure of the accuracy of the detected SNP, centered around the given values. The larger the log-odds ratio is, the more accurate the detected SNP becomes. To understand the reason for the low number of SNPs detected by Bowtie and Novoalign, we carried out

the same experiment while using a quality threshold of 100. The number of highly accurate SNPs increased to 1474 and 1100 for Bowtie and Novoalign, respectively. V8 Hatem et al. Processing of Biological Data BMC Bioinformatics (2013) 14:184 15 Read alignment: conclusion Mapping of short sequences is still subject of active development. Genome indexing tools performed better than read indexing tools. In general, there is no the-best tool among all of the tools; each tool was the-best in certain conditions. Hatem et al. BMC Bioinformatics (2013) 14:184 V8 Processing of Biological Data 16 (2) Variant calling benchmark -> Accurately detecting SNPs is critical e.g. for medical diagnostics. Genome in a Bottle (GIAB) consortium: public-private-academic consortium to develop the technical infrastructure (reference standards, reference methods, and reference data) to enable translation of whole human genome sequencing to clinical practice. GIAB generated a set of highly confident variant calls for one individual in the

1000 Genome project: they integrated 14 variant data sets from 5 NGS technologies, 7 read mappers and 3 variant calling methods, and manually cleaned up discordant data sets. This highly accurate set of SNP and indel genotype calls can be used as gold standard variant genotype data set for systematic comparisons of variant callers. Hwang et al., Scientific Reports 5, 17875 (2015) V8 Processing of Biological Data 17 Variant calling: performance Performance of variant calling pipelines measured by APR for (A) SNP and (B) indels. APR: Area under a precision-recall curve. Hwang et al., Scientific Reports 5, 17875 (2015) V8 Coverage 47 299 x Processing of Biological Data

< 10 x 18 Variant calling: consistency Mean percentage with standard deviation of confidence variant calls with quality 20 for Illumina data sets. -> Generally good agreement (92% overlap of results). For low coverage IonProton data, the overlap is only 15%. Hwang et al., Scientific Reports 5, 17875 (2015) V8 Processing of Biological Data 19 Variant calling: recommendation The authors recommend the use of BWA-MEM and Samtools pipeline for SNP calls and BWA-MEM and GATK-HC pipeline for indel calls. Low coverage data is not suitable for reliable SNP calling. Indels are detected at lower accuracy than SNPs. Hwang et al., Scientific Reports 5, 17875 (2015)

V8 Processing of Biological Data 20 (3) SNPs in 1000 Genomes project The 1000 Genomes Project ran between 2008 and 2015 and created the largest public catalogue of human variation and genotype data up to date. The goal of the 1000 Genomes Project was to find most genetic variants with frequencies of at least 1% in the populations studied. http://www.internationalgenome.org/ V8 Processing of Biological Data 21 Data set We used only the European super-population with 503 individuals and focused on autosomes (chromosomes 1 22). Genes on sex chromosomes X and Y were ignored. We kept autosomal SNPs with a minor allele frequency larger than zero SNP exists allele : variant form of a given gene major allele : most common variant minor allele: second-most common variant We removed: - genes starting with "SNO (small nuclear RNAs) or "MIR ( microRNAs)

- genes with CDS start equal to the CDS end Neininger & Helms, submitted V8 Processing of Biological Data 22 Problem: there exist many overlapping genes Shown is overlap between 3 human genes: MUTH, FLJ13949, and TESK2. Dark boxes : coding sequence. Light boxes : untranslated regions. Veeramachaneni et al. Genome Res. (2004) 14: 280-286 V8 Processing of Biological Data 23 Overlapping genes One could speculate that overlapping genes would be more conserved between species than non-overlapping genes because a mutation in the overlapping region would cause changes in both genes. Then, one would expect that evolutionary selection against these mutations is stronger. However, Veeramachaneni et al. found that this is not the case.

Overlapping human and mouse genes were similarly conserved as nonoverlapping genes. Veeramachaneni et al. Genome Res. (2004) 14: 280-286 V8 Processing of Biological Data 24 How to deal with overlapping genes In the case of overlapping genes, it is problematic to define the genomic regions because they have a different meaning for the 2 overlapping genes. Therefore, we distinguished 2 cases: (1) Overlaps where one gene is located inside another gene. Such genes inside other genes were excluded from the SNP analysis. (2) staggered overlaps (genes overlap partially). We collected all genes with staggered overlap. From each bundle", only one gene was selected randomly to avoid overlapping genes. In total, about 5% of all genes were removed due to overlaps. Neininger & Helms, submitted V8 Processing of Biological Data 25 Refseq

The Reference Sequence (RefSeq) collection at NCBI provides a comprehensive, integrated, non-redundant, well-annotated set of sequences, including genomic DNA, transcripts, and proteins. RefSeq transcript and protein records are generated in different ways: - Computation Eukaryotic Genome Annotation Pipeline Prokaryotic Genome Annotation Pipeline - Manual curation - Propagation from annotated genomes that are submitted to members of the International Nucleotide Sequence Database Collaboration (INSDC) Research question: Are the Single Nucleotide Polymorphism (SNP) frequencies in different genomic regions similar to eachother or not? https://www.ncbi.nlm.nih.gov/refseq/about/ V8 Processing of Biological Data 26 Definition of genomic regions Every gene is located between two intergenic regions. Our definition for these is: First intergenic region : interval between the transcription start site (TSS) of the considered gene and the mid-upstream position between this TSS and the transcription end site (TES) of the closest upstream gene. Second intergenic region : defined analogously according to the TSS of the closest

downstream gene. Intragenic region of a gene : part between its TSS and its TES. Gene promoter : region from 2000 bp upstream to 1000 bp downstream of the TSS. Exons : intervals between the exon start positions and exon end positions (taken from UCSC genome browser). 5' UTRs : exonic segments between the TSS and the CSS 3' UTRs : exonic regions between the CES and the TES. Introns : regions between the exonic gene parts. Neininger & Helms, submitted V8 Processing of Biological Data 27 SNP density in genomic regions Number of SNP variants per kb for different genomic regions. lowest SNP density in coding exons (green) highest SNP density in CpG islands (red, due to frequent deamination of methylated cytosines into thymines) Second-highest SNP density in intergenic regions (grey, low evolutionary

pressure) Neininger & Helms, submitted V8 Processing of Biological Data 28 (4) Isoforms of genes Gene isoforms are mRNAs that are produced from the same locus but are different in their - transcription start sites (TSSs), - protein coding DNA sequences (CDSs) and/or - untranslated regions (UTRs), All these processes may potentially alter gene function. www.wikipedia.org V8 Processing of Biological Data 29 Alternative splicing may affect PP interactions: STIM2 splice variant STIM proteins regulate store-operated calcium entry (SOCE) by sensing Ca2+ concentration in the ER and forming oligomers to trigger Ca2+ entry through plasma membrane-localized Orai1 channels.

Niemeyer and co-workers characterized a STIM2 splice variant which retains an additional 8-AA exon within the region encoding the channelactivating domain. STIM2.1 knockdown increases SOCE in naive CD4+ T cells, whereas knockdown of STIM2.2 decreases SOCE. Overexpression of STIM2.1, but not STIM2.2, decreases SOCE. STIM2.1 interaction with Orai1 is impaired and prevents Orai1 activation. V8 30 Processing of Biological Data Miederer, ..., Lee, ..., Helms, Niemeyer Nature Commun 6, 6899 (2015) Alternative splicing Alternative splicing (AS) of mRNA can generate a wide range of mature RNA transcripts. It is estimated that AS of pre-mRNA occurs in 95% of multi-exon human genes. There is abundant evidence for the expression of multiple transcripts in cells. However, it is less clear whether these transcripts are expressed more or less equally across tissues or whether it would be biologically relevant to designate one transcript per gene as dominant and the rest as alternative. Ezkurdia et al J Proteome Res. (2015) 14: 18801887. V8

Processing of Biological Data 31 Evidence from mRNA expression 3 contrasting large-scale expression studies came to different conclusions. (1) An EST-based study with 13 different tissues predicted that primary tissues generally had a single dominant transcript per gene. (2) In contrast, a large-scale study using RNAseq found that > 75% of proteincoding genes had cell-line-specific dominant transcripts. Those genes with the most splice variants had more dominant transcripts. (3) A second RNAseq study (Illumina Human BodyMap project) found that ca. 50% of the genes expressed in the 16 tissues studied had the same major transcript in all tissues, whereas another third of the genes had major transcripts that were tissue-dependent. One curious result in this study was that the major transcript was noncoding in close to 20% of the protein-coding genes. Ezkurdia et al J Proteome Res. (2015) 14: 18801887. V8 Processing of Biological Data 32 Detect isoforms in proteomic data Ezkurdia et al. performed a re-analysis of 8 HT proteomics MS data sets. At least 2 peptides were detected for 12 716 (63.9%) of the protein-coding genes but alternative protein isoforms only for 246 genes (1.2%).

the vast majority of genes had peptide evidence for just one protein isoform. The isoform with the highest number of peptides was the main proteomics isoform. A unique main proteomics isoform was identified for 5011 genes. Ezkurdia et al J Proteome Res. (2015) 14: 18801887. V8 Processing of Biological Data 33 Comparison proteomics - RNAseq CCDS variants are based on genomic evidence and are variants that are mutually agreed on by teams of manual annotators from NCBI, the Sanger Institute, EBI and UC Santa Cruz. A total of 13 297 genes were annotated with a single CCDS variant. This unique manually curated variant agreed with the main proteomics isoform for 98.6% of the 3331 genes that were compared. APPRIS annotates principal isoforms on the basis of conservation of structure and function and selected a main isoform for 15 172 of the coding genes. Ezkurdia et al. were able to compare the APPRIS principal isoforms and the main proteomics isoforms over 4186 genes. The main proteomics isoform agreed with the isoform with the most conserved protein features for 97.8% of these genes. In contrast, the longest isoform coincided with the main proteomics isoform only for 89.6% of the genes. Ezkurdia et al J Proteome Res. (2015) 14: 18801887. V8 Processing of Biological Data

34 (5) Alternative translation: example TrpV6 channel protein MUSCLE multiple sequence alignment of the The mammalian sequences upstream of the first AUG codon are conserved, but the translated 5-UTR of TRPV6 one from rabbit contains an in-frame stop Identical aa residues (compared with the codon. In contrast, sequences from the other organisms contain several stop human sequence) are shaded; annotated N termini with the first Met+1 are in codons upstream of the annotated AUG and are not conserved. Sequence identity red; is highest among the 40 amino acids * : stop codon in frame upstream of the first Met residue (position +1). This suggests that translation in : gap Fecher-Trost et al. J. Biol. mammals may start at a non-AUG V8 Chem. (2013) 288: 16629 Processing of Biological Data 35

Alternative translation of human TRPV6 Nucleotide alignment of 5-UTR TRPV6 sequences including the AUG triplet encoding the first methionine (red, +1) of the human protein. Red, putative initiation sites; underlined, STOP-codon in frame. Experiments in the Flockerzi group (Medical department, Homburg) showed that translation starts at Thr-40 . Fecher-Trost et al. J. Biol. Chem. (2013) 288: 16629 V8 Processing of Biological Data 36 HT discovery of alternative translation: ribosome profiling Protocol resembles ChIP-Seq. Halt translation by applying ribosome inhibitors. Isolate ribosome-bound mRNAs by size. Then treat sample with a nonspecific nuclease. This results in protected mRNA fragments termed 'footprints'. These ribosome footprints are isolated and converted to a library for deep sequencing. V8

Brar, Weissman, Nature Rev Mol Cell Biol 16, 651664 (2015) Processing of Biological Data 37 PreTIS: predict alternative translation initiation sites Suppose that a ribosome profiling experiment detected 2 start sites for this mRNA sequence: CUG at position -78 and CUG at position -120 (blue colored codons). These start sites are then considered TP start sites. All near-cognate start sites not listed in the ribosome profiling dataset and upstream of the most downstream reported true start site are then considered TN (red colored codons). Light red colored codons : start sites not considered as false starts in the analyses since they are located downstream of the most downstream reported true start site. Grey colored downstream part : annotated CDS sequence Italic (purple) upstream part : -99 upstream window needed to calculate some features. All marked start sites (TP and TN) exhibit a surrounding window of 99 nucleotides as well as a downstream inframe stop codon. In total, this mRNA sequence would provide 2 true start sites and 9 false start sites out of 23 putative starts. V8 Processing of Biological Data Reuter et al Plos Comput Biol (2016) 12: e10005170 38

Data sets used for ML classifier We only included curated mRNA sequences with available mRNA RefSeq identifier (starting with NM_). Raw data is very unbalanced (number of TPs and TNs very different) need to balance data sets (select random TN data points) Reuter et al Plos Comput Biol (2016) 12: e10005170 V8 Processing of Biological Data 39 Flow-chart Data balancing was repeated 10 times to investigate model robustness. Significant features were identified by the Wilcoxon-rank sum test. Reuter et al. Plos Comput Biol (2016) 12: e10005170 V8 Processing of Biological Data

40 Features used Mean value and standard deviation of the 44 features that were used in the best human model. PWM : probability weight matrix Entries of position frequencymatrix (PFM) : sum of occurrences of a nucleotide at position i divided by the total number of sequences contained in S. Reuter et al Plos Comput Biol (2016) 12: e10005170 V8 Processing of Biological Data 41 Evaluation All human models perform very similarly with accuracies of about 80% while the average performance of the mouse model is lower with average accuracies of about 76%,

Reuter et al. Plos Comput Biol (2016) 12: e10005170 V8 Processing of Biological Data 42 Is model transferable to other species? Performance of the best human HEK293 model applied to the mouse ES dataset model is reasonably transferable, suggests universal translation code Reuter et al. Plos Comput Biol (2016) 12: e10005170 V8 Processing of Biological Data 43 Alternative start codons of human gene GIMAP5 AUG at position -203 is a hot

candidate with a very high confidence value of 0.92 of being a true start site. Predicted start sites were subdivided into 4 confidence groups and highlighted by different colors and dashed lines: - very high (best candidates with c 0.9), - high (0.8 c < 0.9), - moderate (0.7 c < 0.8) and - low (t = 0.54 c < 0.7) initiation confidence c. V8 Processing of Biological Data Reuter et al Plos Comput Biol (2016) 12: e10005170 44 Mutation matrix showing the impact of the flanking sequence context of 4 putative start sites of gene GIMAP5 on the predicted initiation

confidence. Virtual SNP analysis of gene GIMAP5 In each case, only one nucleotide is mutated with respect to the reference sequence (top line). Grey : start was predicted as true translational start (predicted initiation confidence > 0.54). white : start was classified as false start. Mutations at the start sites itself were not considered. The numbers reflect the predicted initiation confidence values V8 Reuter et al Plos Comput Biol (2016) 12: e10005170 Processing of Biological Data

45 (6) Removing sequence redundancy Lets assume we want to know whether the amino acid composition of certain protein sequences differs in one genomic region from the other regions. For example, we want to know whether transmembrane (TM) segments of membrane proteins are more hydrophobic than the rest of the protein sequence To check this, we could simply analyze all protein sequences from NCBI, predict the TM segments in them and compare the amino acid compositions. However, this search would likely be biased by - what proteins have been sequenced and which ones not, and - by duplicated sequencing experiments. It is very important to remove sequence redundancy before such analyses! This can be done by software tools such as CDhit or BlastClust V8 Processing of Biological Data 46 BlastClust blastclust -i infile -o outfile -p F -L .9 -b T -S 95 The sequences in "infile" will be clustered and the results will be written to "outfile". The input sequences are identified as nucleotide (-p F); "-p T", or protein. To register a pairwise match two sequences will need to be 95% identical (-S 95) over an area covering 90% of the length (-L .9) of each sequence (-b T) .

https://www.ncbi.nlm.nih.gov/Web/Newsltr/Spring04/blastlab.html V8 Processing of Biological Data 47 Take home messages - Usually one removes sequence redundancy when correlating sequence features with properties of proteins etc. - Check for overlapping genes - Which isoform is relevant? There are substantial differences between what is expressed at the transcript level and what is expressed at the protein level. CCDS and APPRIS appear good resources. - Which translated variant is relevant? May want to try PreTIS Reuter et al. Plos Comput Biol (2016) 12: e10005170 V8 Processing of Biological Data 48

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