In terms of splicing errors, gmap outperformed the other programs by even larger margins. The lengths of the two breaks are indicated above and below the alignment. Because ESTs are of widely differing quality, we assigned each EST a quality score, which was the percentage identity of the EST relative to the genome as determined by the higher identity score between the gmap and blat alignments. We further this work by integrating the detection procedure into the framework of a cDNAgenomic alignment program, and by adding a probabilistic extension that ensures that incorporated microexons are statistically significant. Bioinformatics. We show that Liftoff can accurately map 99.9% of genes between two versions of the human reference genome with an average sequence identity >99.9%. Note that the y-axis is shown on a log scale, as in Figure2. The Author 2005. The NISC Director is responsible for overseeing all aspects of this large, well-respected, and highly productive center that is vital to many research projects at NHGRI and the intramural research programs at many other NIH institutes/centers. We have found it convenient to be able to map and align a single cDNA sequence quickly when needed, and to be able to switch quickly among different genomes or versions of a genome. For the intron case, this path bridges the coordinates for the cDNA sequence but allows genomic coordinates to jump across the intron. The node weights correspond to the exon mismatch penalties (default of 2 per mismatch) and the edge weights are the sum of the exon gap open penalty (2) and gap extension penalty (1). Gmap then uses the mRNA to identify the appropriate genomic segment and to mark it with the coding region and codon positions. First of all, the success of the lift-over is limited by the divergence between the reference and target genomes (Supplementary Fig. The results of this experiment are shown in Table 1. The splicing errors by gmap involved shifted canonical splice sites in 5 sequences, and conversion of semi-canonical (GCAG) splice sites to canonical ones in 4 sequences. On sequence ENST0354373, gmap starts the alignment at position 13, rather than creating an initial exon of 13 nt followed by a non-canonical intron. For each oligomer size, counts of all overlapping oligomers in the masked part of the human genome are shown by the top line, and the counts of distinct oligomers are shown by the topmost sigmoid line. All rights reserved. The resulted layouts are expected to correspond to the unique regions of the target genome, thus the uncertainty caused by repetitive regions is minimized at this stage. As was done with the GRCh37 to GRCh38 lift-over, we compared the gene order in GRCh38 to that in PTRv2 and found 2477 genes in PTRv2 to be in a different relative position. Alignment 1 (green) has 4 gapless blocks containing exons 14 which are represented by nodes AD in the graph. Because access to files is much slower than to memory, our file-based strategy is enabled by a minimal sampling strategy that attempts to perform as few oligomer lookups as possible, while still mapping reliably to an entire genome. Likewise, our knowledge of microexons and chromosomal rearrangements can be enhanced by accurate prediction of gene structures and chimeric ESTs. At the beginning of the left loop (), the reads are mapped to the assistant genome, and only the uniquely mapped (UM) reads are kept.The UM reads are further filtered by two uniqueness rules (Section 2.2). Histogram showing the distribution of exon sequence identity of protein-coding and lncRNA genes in GRCh37 and GRCh38. 63 of these genes mapped end-to-end with Liftoff (Supplementary Table S2) and 27 mapped partially with an alignment coverage less than 100% but greater than the 50% threshold mentioned above. We attempted to map all protein-coding genes and lncRNAs on primary chromosomes (excluding alternative scaffolds) in the GENCODE v19 annotation (Harrow et al., 2012) from GRCh37 to GRCh38. The compressed format allocates 3 bits per position, allowing for representation of A, C, G, T, N and X. The definition of same location depends upon the length of the query cDNA, with an allowed genomic expansion of 1000 times the query length, subject to a default upper limit of 1 million nucleotides. Rather than attempting to fix an existing approximate alignment, the method computes the whole subalignment in the region surrounding an intron. 2011;27:1481-8. While these parameters work well for the examples presented here, Liftoff allows the user to change or add any additional Minimap2 options. u and v are on the same chromosome or contig. We consider a gene to be successfully mapped if at least 50% of the reference gene maps to the target assembly. The most well-known example of this is the human genome, but other model organisms such as mouse, zebrafish (Church et al., 2011), rhesus macaque (He et al., 2019), maize (Jiao et al., 2017) and many others have had a series of gradually improved assemblies. To visualize the co-linearity of the gene order between the two assemblies, we plotted each gene as a single point on a 2D plot where the X coordinate is the ordinal position of the gene in GRCh37 and the Y coordinate is the ordinal position in GRCh38 (Fig. The top plot shows the number of ESTs at each quality level. Therefore, we have built into gmap the ability to map and align a given cDNA over multiple strains simultaneously. 2021) involves the imaging of fluorescently labeled DNA molecules and their alignment to reference genome sequences. There are some limitations with annotating new assemblies using a lift-over strategy rather than de novo. By comparison, other programs had error rates of 6.356.7%, due predominantly to shifted canonical splice sites. First, gmap uses an oligomer index table for genomic mapping. The results of our comparison are shown in Figure 6. Rather than annotating genomes de novo, we can take advantage of the extensive work that has gone into creating reference annotations for many well-studied species. In the gold standard, we found 2 non-canonical introns that could be converted to a canonical one with 0 substitutions or gaps; 38 that could be converted with 1 substitution or gap; 6 with 2 substitutions or gaps; 11 with 3 substitutions or gaps; and 3 with 4 substitutions or gaps. The tool contains functions such as genome annotation, feature extraction etc. The fraction of such short breaks relative to the total alignment length is defined to be the defect rate, and is used to classify the cDNA sequence as being of high (defect rate <0.3%), medium (0.31.4%), or low quality (>1.4%). Alignments 2 (purple) and 3 (orange) each have 1 gapless block containing exon 5 represented by nodes E and F respectively. Gmap reported two 5 exons not reported by GeneSeqer, with lengths of 9 and 8 nt. The preference for trinucleotide gaps reflects selection pressure at the protein level to avoid frameshifts and preserve the coding region. The sufficiency limit has a default value of 60, which expresses our calculated expectation that we should find at least one matching 8-mer between the cDNA and genome within that distance, even accounting for extremely low sequence quality. (, Oxford University Press is a department of the University of Oxford. Therefore, to decide whether a short exon does indeed exist, the algorithm attempts to align the region under the two assumptions that the short exon is present (meaning two introns and a middle exon) or that it is absent (meaning one intron). UCSC liftOver failed to map 125 genes. The existence of short exons is resolved by an exon testing procedure that compares alignments with and without the short exon. Using the coordinates of the aligned blocks in the shortest path, the coordinates of each exon are converted to their respective coordinates in the target genome. Moreover, the ability of an alignment program to detect splice sites in the presence of sequence error and in the absence of prior bias may alter our assessment of the frequencies of canonical, semi-canonical and non-canonical splice sites. However, for the second EST, which has two sequence differences, only gmap and sim4 can recognize the canonical intron. The distance from the start of u to the end of v in the target genome is no greater than 2 times that in the reference genome. Our data set contained a total of 8634 exons. In addition to introns, other types of sequence differences can cause 8-mers not to align in the oligomer chaining procedure and thereby yield discontinuities or jumps in the cDNA or genomic coordinates in the alignment. Tomato (Solanum lycopersicum), an important horticultural crop, is an ideal model species for the study of fruit development. In order to reduce the complexity to O(mg2), we impose a sufficiency limit on the look backward. Likewise, among all 14-mers, only 22.5% specify a unique position in the genome. These regions between exons are annotated as introns, although some programs may annotate these simply as small cDNA deletions. . These alignments represent all splicing differences between the two programs on a dataset of 5000 Arabidopsis cDNAs. In addition, intron lengths have significantly different distributions in different species, with Caenorhabditis elegans, Drosophila melanogaster and A.thaliana having shorter intron lengths on average than Saccharomyces cerevisiae and human beings, and lower organisms only rarely having introns of the 1000-nt or longer variety found commonly in higher eukaryotes (Lim and Burge, 2001). The total space of possible oligomers increases exponentially, as shown by the exponentially increasing line. Thomas D. Wu , Colin K. Watanabe, GMAP: a genomic mapping and alignment program for mRNA and EST sequences, Bioinformatics, Volume 21, Issue 9, May 2005, Pages 18591875, https://doi.org/10.1093/bioinformatics/bti310. For the human genome, genomic oligomer files require a total of 1.9 GB and the genomic sequence file requires 1.1 GB compressed (3.1 GB uncompressed). The calculation imposes a higher minimum length requirement for a microexon in a longer intron, to offset its higher likelihood of an exact match by chance. Heinz 1706 that was 799.09 Mb in length, containing 34,384 predicted protein-coding genes and 65.66% . Such an approach is relatively sound because oligomer chaining bounds the solution well from a global perspective, leaving only small sequence edits to be performed. In other cases, differences between the two genomes cause the gene to align in many fragmented pieces, and the optimal mapping is some combination of alignments. In this process, exons are not created explicitly, but instead emerge implicitly from the globally optimal distribution of 8-mer matches between the cDNA and genomic segment. BioProject (formerly Genome Project) A collection of genomics, functional genomics, and genetics studies and links to their resulting datasets. The sampling process begins by scanning both ends of the cDNA sequence, and monitoring the results until a pair of 24-mers match to approximately the same location in the genome. When multithreading is enabled, one thread handles reading of the input, one handles writing of the output alignments, and one or more worker threads each processes an individual cDNA sequence. We attempted to map all protein-coding genes on chromosomes 1-22 and chromosome X in the GENCODE v33 annotation (Frankish et al., 2019) from GRCh38 to an assembly of the chimpanzee (Pan troglodytes), PTRv2 (GenBank accession GCA_002880755.3). Note that this limit applies only to the cDNA sequence coordinates; there is no limitation on the look backward in genomic sequence coordinates. Splicing errors. Gmap has a mode where a set of ESTs can be aligned relative to a reference sequence. To compare Liftoff to an existing commonly used method, we lifted over genes between the same 2 assemblies using the UCSC liftOver tool. Although one advantage of an integrated mapping and alignment program over separate programs is convenience, coupling of the mapping and alignment tasks also provides functional advantages. In the fourth and final pass, the algorithm extends the 5 and 3 ends of the cDNA sequence, by using DP for the sequence ends. The long oligomer approach is exemplified by MGAlign (Ranganathan et al., 2003): although it does not perform mapping on a genomic scale, it initially aligns a cDNA to a given genomic segment by scanning 20-mers from the ends of the cDNA. Interestingly, 9 out of the 10 blat splicing errors were associated with microexons, because they were missed, predicted with the wrong length, or matched to the wrong place in the intron. The program maps and aligns a single sequence with minimal startup time and memory requirements, and provides fast batch processing of large sequence sets. On a set of Arabidopsis cDNAs, gmap performed comparably with GeneSeqer. In addition, there were 3472 ESTs (or 7%) that had 60% identity or less by both programs. If we consider the 20,635 ESTs with 98% identity or more, 18,363 (89.0%) were ties, 1953 (9.5%) favored gmap, and 319 (1.5%) favored blat. In that time, the program has undergone continual evolution, both to improve its accuracy and speed, and to provide additional functionality. We compared gmap with blat, the only other integrated program for mapping and alignment available to us. We show that Liftoff can accurately map 99.9% of genes between two versions of the human reference genome with an average sequence identity >99.9%. Currently, there are 13420 eukaryotic genome assemblies in GenBank, of which 10000 have been added in the last 5years alone. On the other hand, if there are a large number of candidate genomic locations, then gmap begins a sampling process that uses information from the middle of the cDNA sequence. Specifically, we describe: (1) a minimal sampling strategy for genomic mapping, (2) oligomer chaining for generating approximate gene structures, (3) sandwich DP for identifying splice sites, and (4) microexon identification with statistical significance testing. Methods: A high-risk pregnant woman identified at the Prenatal Diagnosis Center of Hangzhou Women's Hospital in October 2021 and her family members were selected as the study subjects. The 4 largest of these inversions are on 4, 5, 12 and 17 (Soto et al., 2020) hence their visibility at this scale. We demonstrate that this approach can map more genes than sequence homology-based approaches. Running times for the Arabidopsis data set were 42 min for GeneSeqer and 1 min for gmap; these times are not entirely comparable, because GeneSeqer needed to be restarted for each cDNAgenomic alignment. The score for the cell is the score of the previous cell plus 1 to indicate the length of the chain. Genomics is the study of genomes. This revealed 361 genes (1.3%) in a different relative position in GRCh38 compared to GRCh37. The first column shows a canonical intron (marked by >) from an EST with one sequence difference nearby: a single gap (marked by ). The remaining 16 cases involve differences in splice sites and one microexon. To find the optimal path, each matrix is scored outside in by the usual Needleman and Wunsch (1970) procedure, which enforces an alignment to the ends of the intron. Existing programs for cDNAgenomic mapping and alignment, cited in the Introduction, provide a foundation for further advances. We excluded these introns from being counted as either shifting or overcalling errors, since many programs are designed to convert these non-canonical introns into canonical ones. Figure 4 shows some cases of splice site detection in the presence of sequence error. The two matrices are solved outside in, as shown by the direction of the arrows. We tested all programs on an Intel Linux machine with 2 Xeon processors at 2.4 GHz with 2 GB of RAM running RedHat Linux. Previously, we have used Liftoff to map genes from GRCh38 to a new Ashkenazi human reference genome (Shumate etal., 2020). Repeating this costly process for each updated or new genome assembly is unnecessary. Genetic maps are species-specific and comprised of genomic markers and/or genes and the genetic distance between each marker. Also, our procedure looks only within 12 nt of the alignment boundaries rather than the 30 nt by Volfovsky, because longer microexons would have been identified by oligomer chaining. If a given 24-mer has a match somewhere in the genome, an entry for the 24-mer can be found in the expected hash bin. Gmap had gene structure errors in 9 sequences, and splicing errors in 9 sequences. Gmap is capable of finding and reporting chimeras, or ESTs whose 5 and 3 ends map to different genomic regions. The running time for dds/gap2 is extremely long, which probably reflects its reliance upon alignment procedures at the nucleotide level. We also found a full-length sequence AAC50956 in the patent database that GeneSeqer aligns to give the same single 630-nt intron as gmap. These linked lists are represented in the figure as a vertical stack of cells at each cDNA position. Existing approaches to splice site identification are based upon two ideas. Background Genetic and functional genomics studies require a high-quality genome assembly. Oxford University Press is a department of the University of Oxford. The bottom graph of Figure 6 shows the non-overlapping cases, which include the 1206 ESTs aligned to different genomic locations and the 356 aligned by only one program. Instead of pre-loading the entire oligomer index file into memory, gmap looks up oligomers as needed directly from the file. Results of aligning 883 Ensembl mRNAs from chromosome 22. Availability: Source code for gmap and associated programs is available at http://www.gene.com/share/gmap, Supplementary information: http://www.gene.com/share/gmap. In two of these cases, AY086916 and AY87013, marked in Figure 7 with an (I), the genomic splice site predictions of the two programs are identical, and the differences lie only in the predicted exonexon boundary. The lift-over procedure is repeated until all valid mappings have been found. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide, This PDF is available to Subscribers Only. Another feature unique to Liftoff is the option to find additional copies of genes in the target assembly not annotated in the reference. We recently used Liftoff with this feature enabled to annotate our improved assembly of the bread wheat genome, which contains 15.07 gigabases of anchored sequence compared to 13.84 in a previous reference genome (Alonge et al., 2020). In one study by Marqus Bonet and others, the genomes of the 233 primate species were used to classify 4.3 million common gene variants present in the human genome 2. The determination of linear order with which genetic units are arranged with respect to one another (gene order). To whom correspondence should be addressed. Genomic alignment programs require the user to supply the correct genomic segment to align to, but the correct segment may not be apparent when there are multiple candidate genomic locations. Once a genome is processed by gmap_setup, the user may retrieve arbitrary segments from the genome using the auxiliary program get-genome. This solution corresponds to an intron in EST sequence BF846255, shown in the middle of Figure 4. Source code and documentation for gmap and associated programs are available for open use at http://www.gene.com/share/gmap. Accurate and fast genomic mapping and alignment should facilitate our exploration of the genome and our understanding of the structure, function and evolution of genes. Mapping over multiple strains requires that we augment our genomic index table with 24-mers from all strains. Microexons as short as 1 nucleotide in length have found apparent experimental support (McAllister et al., 1992; Sterner and Berget, 1993; Simpson et al., 2000; Carlo et al., 2000), and a computational study suggests that between 0.5 and 1.6% of mRNA sequences in various species contain microexons (Volfovsky et al., 2003). F, Lapuk A, et al. The compression scheme stores each block of 32 nucleotides into three 32-bit words, with the first two words holding the first two bits of each nucleotide, and the last word holding the third bit (which is set only for non-ACGT letters). Here, we describe Liftoff, a new genome annotation lift-over tool capable of mapping genes between two assemblies of the same or closely related species. This entry then provides the appropriate offset into the position file. These jumps are resolved by various nucleotide-level alignment procedures, represented in the bottom of the figure by various DP matrices. This alignment may contain jumps in cDNA or genomic coordinates, due to introns, cDNA insertions or sequence differences. Gmap compares the translation of each EST against the translation of the reference sequence to report a summary of protein sequence variations, including SNPs, amino acid insertions and deletions, and alternative splice forms. The Minimap2 parameters are set to output up to 50 secondary alignments for each sequence in SAM format. The seed-and-extend strategy is found in a variety of programs, including those for genomegenome alignment (Chain et al., 2003; Morgenstern, 1999; Batzoglou et al., 2000; Kent and Zahler, 2000; Schwartz et al., 2000; Ma et al., 2002; Brudno et al., 2003a; 2003b; Bray et al., 2003; Kalafus et al., 2004), and constitutes the approach in several cDNAgenomic alignment programs. Before gmap can handle a given genome, it requires that the genome be pre-processed, by constructing a genomic oligomer index (consisting of an offset file and a position file) and a genomic sequence file. In addition to using highly specific 24-mers, gmap employs an adaptive sampling scheme designed to utilize mapping information from different parts of the cDNA sequence. For each gene, Liftoff finds the alignments of the exons that maximize sequence identity while preserving the transcript and gene structure. BSMAP combines genome hashing and bitwise masking to achieve fast and accurate bisulfite mapping. In this case, we found other sequences with an additional 462 nucleotides relative to the test cDNA, which gmap introduces as a new middle exon and which GeneSeqer appends to its existing middle exon. Note that differences in the genome sequences themselves may result in Liftoff mapping a gene to a paralogous location. A third mode of gmap allows the user to provide both a genomic segment and one or more cDNA sequences. Sandwich DP is one of several nucleotide-level alignment procedures used to fill in gaps in the approximate alignment. MGAlign also applies DP, both to extend its fragments and to combine local alignments into longer ones. Tags allow one to mark and retrieve subsets of intervals. Eukaryotic genome annotation is a challenging, imperfect process that requires a combination of computational predictions, experimental validation and manual curation. For AY08166, GeneSeqer makes an alignment on the wrong strand. In the remainder of the paper, we review existing work on cDNAgenomic mapping and alignment, and describe the methods underlying gmap. Therefore, the original nucleotide may have been resubstituted in the given position, resulting in no change. Sandwich DP involves two DP matrices, one for each end of an intron, and attempts to find the best alignment path across the diagonals of both matrices. In sandwich DP, the goal is to find an optimal path from the upper left corner to the lower right corner. In addition, some inter-exon regions were also extremely short, with 125 having lengths of 17 nt. To evaluate the robustness of the two programs to sequence error, we created mutated data sets at rates of 1 and 3%, using the same approach as in Experiment 1. The main goal of Liftoff is to align gene features from a reference genome to a target genome and use the alignment(s) to optimally convert the coordinates of each exon. Gmap can be run on a fasta file containing one or more cDNA sequences. Next we provide examples of how these methods in gmap lead to improved splice site and gene structure prediction. In experiment 3, we evaluate the performance of gmap on another species, namely, the plant Arabidopsis thaliana. The use of 18-mers can give additional sensitivity for divergent sequences, such as in the cross-species genomic mapping of mouse cDNAs onto the human genome, and vice versa. Gene mapping is the sequential allocation of loci to a relative position on a chromosome. In contrast, gmap handles this problem by using a formal DP procedure that we call sandwich DP. Mitsiades and colleagues utilize functional genomics data in over 700 cancer cell lines, to identify genes with preferentially essential functions in multiple myeloma, which may represent targets . We again compared our results to UCSC liftOver. The genomic codon boundaries also enable gmap to perform a frameshift-tolerant translation of the EST. Each comparison shows the GeneSeqer alignment on top and the gmap alignment on bottom. This is seen as the third EST, in which Sim4 appears to overcall a canonical intron by introducing gaps of 5 nt in an mRNA that otherwise has perfect sequence identity to the genome. The startup time for the standalone version of blat is several minutes, which makes it inconvenient for a researcher who wishes to map a single cDNA sequence to a genome, or who wishes to switch quickly among different genomes or versions of a genome. The gene order appears perfectly co-linear; however, there are some exceptions not visible at the scale of the whole genome. Then we compare the performance of gmap with existing programs in three large-scale experiments. This figure shows alignments generated by various programs around introns in three sequences. Actually, sandwich DP can be used to handle not only introns, but also long cDNA insertions relative to the genome, which occur rarely. Chimpanzee Sequencing and Analysis Consortium. In additional previous work, we used Liftoff to annotate an updated assembly of the bread wheat genome, Triticum aestivum (Alonge et al., 2020). In this mode, the user provides gmap with both a full-length mRNA and a fasta file of ESTs. Search for other works by this author on: Department of Computer Science, Johns Hopkins University, Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Nodes in the graph are weighted according to mismatches within exons. Programs that do provide integrated mapping and alignment, namely, blat and squall, are intended primarily for batch or server mode, not for single query or interactive use. Node E is not on the same strand as alignments 1 and 2 and is therefore only connected to the start and end. Such short exons pose an acknowledged problem for cDNAgenomic alignment programs (Florea et al., 1998). Sequencing errors are especially prevalent in ESTs, where error rates are estimated to be 1.5% for high-quality sequences (Zhuo et al., 2003) and 34% overall (Richterich, 1998). Log scale used to make the counts of just 1 or 2 genes visible; all bins below 97% identity contain at most 4 genes. We anticipate that Liftoff will be a valuable tool in improving our understanding of the biological function of the large and rapidly growing number of sequenced genomes. Run times are in (hours:minutes:seconds) are for mapping and alignment by GMAP and BLAT, and for alignment by the other programs. We also note that MGAlign shows a substantial increase in running time with the mutated data sets, perhaps reflecting some underlying characteristic of its handling of substitutions and gaps. The methods employed by gmap enable it to handle certain types of alignment problems that pose challenges for existing programs. [1] Gene mapping describes the methods used to identify the location of a gene and the distances between genes. We also show that Liftoff can map genes across species by successfully lifting over 98.3% of human protein-coding genes to a chimpanzee genome assembly with 98.2% sequence identity. In order to reduce the complexity further to O(mg), we note that one cell in the linked list for a given 8-mer usually has a score that dominates over the scores of other cells in the list. This mode gives gmap the same functionality as pure genomic alignment programs, and is useful for computing cDNA alignments on the fly for a particular genomic region of interest. The compressed format stores only differences relative to the genomic sequence. This process is repeated until there are no genes mapped to overlapping loci. However, a cDNA sequence may have a local concentration of mismatches or gaps that precludes 8-mers from being identified in a particular stretch. To calculate the number of genes out of order in GRCh38 with respect to GRCh37, we calculated the edit distance between the gene order in each assembly. Because this offset file contains an entry for each possible oligomer, its size grows exponentially with the oligomer length. Key: Mis5, Mis3, MisM and MisI missing 5, 3 microexons, and other internal exons; Extr = extra exon; Shift = shifted canonical intron to another genomic position; Over = overcalled canonical intron; Mult = multiple errors of a given class. Diagram showing the steps taken by Liftoff when mapping human transcript ENST00000598723.5 to the chimpanzee (PTRv2) homolog on chromosome 19. Using pyfaidx (Shirley et al., 2015), Liftoff extracts gene sequences from the reference genome and then invokes Minimap2 to align the entire gene sequence including exons and introns to the target. The procedure is illustrated in the top part of Figure 2. The results of the two programs were roughly equivalent. However, the cDNAgenomic alignment problem is important enough to warrant programs specialized for the task. Comrad: detection of expressed rearrangements by integrated analysis of RNA-Seq and low coverage genome sequence data. Indel analysis has thus become one of the most common practices in the lab to evaluate DNA editing events generated by CRISPR/Cas. Blat breaks the cDNA into 500-bp chunks, uses these chunks to create alignment fragments through a recursive seed-and-extend method, and then uses DP to stitch together these subalignments. The average sequence identity in exons of successfully mapped genes was 98.21% (Fig. With that in mind, we have developed sRNAPipe, a pipeline to perform successive steps of small RNA mapping, counting, normalization, drawing publication-quality figures by plotting . Several indel analysis tools have been reported, however, it is often required that users . A similar mutation paradigm has been used to evaluate ab initio gene structure prediction programs (Burset and Guig, 1996). In this tutorial we'll run some common mapping tools on TACC. It can be compiled and run on any modern Unix system with a 32-bit or higher architecture. This pre-scan prevents unnecessary work later, because most of the 8-mers in the longer genomic sequence are irrelevant. Sequence ENST0355936 was placed by gmap on chromosome 2 and by blat on chromosome 7, and sequence ENST0357004 was placed by gmap on chromosome 1 and was not localized by blat to anywhere on the human genome. Gmap has an explicit procedure for finding microexons, based on the method by Volfovsky et al. This per-codon penalty is equal for gaps of 1, 2 and 3 nucleotides, likewise for 4, 5 and 6 nucleotides, and so on. We found that UCSC liftOver failed to map 597 genes. We also showed that we could lift-over nearly all protein-coding genes from GRCh38 to the chimpanzee genome, PTRv2, with an average sequence identity of 98.2%. Results Here, we assembled an updated reference genome of S. lycopersicum cv. Genomic mapping can be accomplished rapidly because of the near-identity between a cDNA sequence and its corresponding genomic exons, which manifests as regions of exact matches. For each candidate cluster of 24-mers, the program extracts the corresponding segment from the genome, with the correct strand of the genome determined by the orientation of the matching 24-mers. In addition, the NISC Director serves as an important advisor and consultant on all aspects related to . Difficulties generally arise when a cDNA sequence differs from its corresponding genomic exons, due to polymorphisms, mutations or sequencing errors. When these conditions are met, the program calculates a lower bound on the microexon length that satisfies a given statistical significance level (p < 0.01 by default). If the match is spurious, a better alignment should result by splitting the short exon and merging the halves into adjacent exons. Although this representation facilitates the insertion, deletion and substitution of subalignments, it can cause contention for heap memory when multiple threads need to build up their linked lists simultaneously. Also, we require that a microexon be reported only if it matches perfectly to the genomic sequence and is surrounded by two canonical introns. In terms of microexons, gmap identified all 25 microexons in the gold standard that were not adjacent to a short intron. The third example in Figure 5 shows how initial and terminal exons can be difficult for some alignment programs to find. Sandwich DP for identifying splice site boundaries. Therefore, gmap has dedicated memory allocation procedures that give each thread its own pool of heap memory as needed, thereby minimizing heap contention. Blat requires a somewhat longer time to start its server.) Annotations were added to indicate whether the corresponding introns had a canonical or non-canonical pair of dinucleotides, and to mark short exons (10 or fewer nucleotides) and short introns (7 or fewer nucleotides). All rights reserved. Existing programs exploit this fact either by finding clusters of relatively short oligomers, such as 11-mers (blat) or 14-mers (ssaha and squall), or by using fewer long oligomers. Such models are used widely in ab initio gene finding programs (Uberbacher and Mural, 1991; Burge and Karlin, 1997; Lukashin and Borodovsky, 1998; Salzberg et al., 1999; Reese et al., 2000) and in homology-based gene finding programs (Guig et al., 1992; Huang et al., 1997; Gotoh, 1999; Batzoglou et al., 2000; Korf et al., 2001; Novichkov et al., 2001; Rinner and Morgenstern, 2002; Brendel et al., 2004), and their use has continued in many cDNAgenomic alignment programs. Gene structure errors occurred in cases where the genomic alignment program missed one or more 5, 3 or internal exons (either microexons or longer ones), or inserted an extra exon. Extending this indexing scheme to 24-mers would yield a sparse offset file of 424 = 281 trillion 32-bit entries, which would be prohibitively large to store. In addition to successfully mapping 100839 of the 105200 reference genes to this large and complex genome, we found 5799 additional gene copies using a strict sequence identity threshold of 100%. Our DP procedures allow us to handle codon insertions and deletions gracefully by an appropriate gap penalty function. In addition, the source code for Liftoff is available at https://github.com/agshumate/Liftoff. Although a full discussion of these features is beyond the scope of this paper, we mention them briefly here. It may be stored in a compressed format, which facilitates the reading of the entire genome into RAM, when sufficient RAM is available. Here, in addition to describing the algorithm itself, we present two more examples demonstrating the accuracy and versatility of Liftoff. Results: On a set of human messenger RNAs with random mutations at a 1 and 3% rate, gmap identified all splice sites accurately in over 99.3% of the sequences, which was one-tenth the error rate of existing programs. Run time for the test set was 3 h and 2 min for blat and 32 min for gmap. For Permissions, please email: https://doi.org/10.1093/bioinformatics/bti310, Receive exclusive offers and updates from Oxford Academic, Autopsy Pathologist and CLIA Medical Director Leadership Opportunity University of Vermont Health Network, MEDICAL MICROBIOLOGY AND CLINICAL LABORATORY MEDICINE PHYSICIAN, CLINICAL CHEMISTRY LABORATORY MEDICINE PHYSICIAN. Next, to demonstrate a cross-species lift over, we map protein-coding genes from the human reference genome to a chimpanzee genome assembly. Sampling terminates when the correct genome location is resolved to a limited number of good candidates. Observations of nucleotide frequencies around splice sites indicate that they are species-specific (Senapathy et al., 1990). In our institution, we are able to map and align the GenBank set of approximately 6 million human ESTs onto the genome using a single computer with three worker threads in less than 2 days. End sequence alignments are computed by constraining one end of the alignment and allowing the distal end to terminate at an optimal stopping point. We used gmap to map and align the cDNAs to the Arabidopsis genome (The Arabidopsis Genome Initiative, 2000). Some of these ordinal differences are visible at the whole-genome scale (Fig. In this paper, we introduce an integrated genomic mapping and alignment program called gmap (Genomic Mapping and Alignment Program). First, we map genes between two versions of the human reference genome. Running times for the remaining programs measure cDNA alignment to their corresponding genomic segments, and include the time needed to restart the program for each alignment. Each position in the array corresponds to an overlapping 8-mer in the cDNA sequence, and each 8-mer has a linked list of positions in the genomic segment where that 8-mer is found. N. Alachiotis, A. Stamatakis, P. Pavlidis, OmegaPlus: A scalable tool for rapid detection of selective sweeps in whole-genome datasets. To help determine which program gives the correct result, we looked for supporting evidence from other ESTs or mRNAs that map to the splice site. Some exons were extremely short, with 41 exons having lengths of 310 nt. Introns have characteristic patterns at their splice sites, which cDNAgenomic alignment programs must take into account. Gmap has the capability of looking up information in a genomic map file to find information relative to a given cDNA alignment. (In addition, some 32-bit machines limit file offsets to 2 GB, which makes compression necessary on these machines for random file access functions to work properly.) The mismatch, gap open and gap extend parameters can be changed by the user. Instead, GeneSeqer extends the previous exon through a stretch of 2 gaps and 14 mismatches. The annotations in our genomic map files can be of arbitrary length, meaning that one may store sequences, entire alignments, or other arbitrary genomic bounds such as cytogenic bands and syntenic regions. (2003) and applied in a large-scale study. Accordingly, cDNAgenomic alignment programs may potentially perform differently on different species. Finally, we describe the implementation of gmap and additional features provided by the program. To find this combination, Liftoff uses networkx (https://github.com/networkx/networkx) to build a directed acyclic graph representing the alignments as follows. The compressed alignments may be uncompressed to their original form using a provided utility program. The program generates accurate gene structures, even in the presence of substantial polymorphisms and sequence errors, without using probabilistic splice site models. Furthermore, some recent experimental results have confirmed some novel chimeras detected using genomic alignments of ESTs (Hahn et al., 2004). These procedures are applied in a particular order in four passes through the alignment. Supporting evidence was found for five cases, marked in Figure 7 with an (E). Times for gmap and blat do not include startup time for the server or for memory mapping of the oligomer index files. In terms of gene structure, it had two differences from the gold standard, for a per-sequence error rate of 0.2%. This gene has a large intronic deletion in PTRv2 and does not have and end-to-end alignment, but it can still be successfully lifted over using our algorithm. In the first pass, the algorithm solves regions where the cDNA and genomic coordinate jumps are approximately equal, indicating the presence of small sequence differences such as mismatches or short insertions or deletions. Minimap2 produces 3 partial alignments of this gene to PTRv2. Other programs have been developed to align a cDNA to a given genomic segment, including est_genome (Mott, 1997), dds/gap2 (Huang, 1996), sim4 (Florea et al., 1998), Spidey (Wheelan et al., 2001), GeneSeqer (Usuka et al., 2000; Schlueter et al., 2003) and MGAlign (Lee et al., 2003; Ranganathan et al., 2003). Unlike current coordinate lift-over strategies which only consider sequence homology, Liftoff considers the constraints between exons of the same gene and constraint that distinct genes need to map to distinct locations. Improvements in DNA sequencing technology and computational methods have led to a substantial increase in the creation of high-quality genome assemblies of many species. Second, the annotation of the new assembly will only be as complete as the reference. The advent of genomics and the ensuing explosion of sequence . We have found that the use of global information is particularly important in the presence of sequence polymorphisms or errors, which can adversely affect local decision-making for extending fragments. The running time for gmap was for a single thread. Supplementary data are available at Bioinformatics online. Among the 43,407 overlapping cases, 32,187 (or 74.2%) ESTs had a tie score; 8032 cases (18.5%) had a better alignment by gmap; and 3188 cases (7.3%) had a better alignment by blat. For genomic mapping, gmap uses a sampling strategy designed to minimize the number of oligomer lookups needed to map a cDNA reliably to the genome. Published by Oxford University Press. Our sampling strategy involves more than scanning long oligomers from the ends of a cDNA to find a matching pair. Out of 19878 genes, we were able to map 19543 (98.31%). The array of linked lists is generated by first pre-scanning the cDNA for overlapping 8-mers and noting which 8-mers are present, and hence relevant. This classification enables appropriate parameters for nucleotide-level alignment to be selected automatically, so that substitutions and gaps are more likely to be introduced for low-quality sequences, and less likely for high-quality sequences. To extend the genomic segment to regions that may be relevant for further alignment at the oligomer and nucleotide level, the program looks up the genomic positions of the nearest 12-mers that match to the ends of the cDNA sequence. Notation is the same as in Figure 4, with the addition of the ] character to indicate an ATAC intron, and a compressed view of exons for AY086965. Here, we demonstrate Liftoffs ability to lift an annotation to an updated reference genome by lifting genes from the two most recent versions of the human reference genome, GRCh37 and GRCh38. The size of the position file is determined by the genome size and by how often oligomers are sampled in the genome. The third column shows a non-canonical intron (marked by =) with no mismatches or gaps. Because the two error classes are not mutually exclusive, we also tallied the union of sequences with one or more errors of any type. Because introns will cause 8-mers in the cDNA not to match, the algorithm compensates for such cases by adding enough points to ensure that local extension does not gain an unwarranted advantage over an intron. Potential microexons are then scanned across the intron using Boyer and Moore (1977) sublinear-time string matching, and accepted if they are surrounded by the requisite AG and GT dinucleotide pairs. For example, Spidey and MGAlign search for splice sites in the overlap between adjacent exons, and then trim the exons at the highest-scoring splice site, whereas sim4 has an intron shifting procedure that adjusts the exonexon junction to find the best pair of splice sites. The graph shows that below a quality score of 85%, alignment quality was evenly divided between gmap and blat. In the Algorithm section, we provide a detailed description of the specific methods underlying gmap; in the rest of this section, we summarize the basic similarities and differences of our methods relative to existing ones. Genomic studies are characterized by simultaneous analysis of a large number of genes using automated data gathering tools. Edges are assigned a weight according to the length of gaps within exons. For each cell, the DP procedure looks for an optimal previous cell, as represented by thin diagonal lines between cells. Comparing the gene order revealed 4 large regions on the homologs of chromosomes 4, 5, 12 and 17 where the gene order is inverted. Instead, a more scalable approach is to take the annotation from a previously annotated member of the same or closely related species, and then map or lift over gene models from the annotated genome onto the new assembly. The alignments for these cases are shown in Figure 7. GRCh38 fixed a number of mis-assemblies and single base errors present in GRCh37 (Guo et al., 2017), so it is expected that the gene sequence and order are not entirely identical. This translation maximizes the amount of EST information available to identify putative point mutations and polymorphisms, but of course misses potentially true frameshift mutations that may lead to a premature stop codon. In turn, heap contention prevents multithreading from using the full potential of multiple parallel processors. Such sequencing errors, especially near exonexon junctions, can complicate the detection of splice sites. For approximate alignment, oligomer chaining attempts to find a path of 8-mers that match between the cDNA sequence and each genomic segment found in the mapping step. One cost of our approach is greater computational complexity than one based on larger fragments. In this section, we discuss the methods used by gmap in the context of each of the major components needed for cDNAgenomic mapping and alignment. Two nodes u and v are connected by an edge if the following conditions are true. However, each release of a genome needs to be set up only once, and the resulting binary files are portable across different computer architectures, because gmap translates the file contents as necessary for big-endian and little-endian platforms. To disregard minor differences between alignments, if the difference between alignment scores was 10 points or less, we considered the alignments to be a tie. By default, opening a gap in an exon incurs a penalty of two, and extending it incurs a penalty of one. Another issue in our development work has been computational speed, both for processing a single cDNA and for processing a large batch of ESTs. The STS concept was introduced by Olson et al (1989). 5) including 4 large regions on the chimpanzee homologues of chromosomes 4, 5, 12 and 17 where the gene order is inverted due to large-scale chromosomal inversions. IITs permit retrieval of all k overlapping intervals for a given query interval in O(k + log2n), where n is the total number of intervals in the database. Mapping and alignment of cDNA sequencesboth messenger RNAs (mRNAs) and expressed sequence tags (ESTs)onto the genome has become a central procedure in genome research. After building this data structure, oligomer chaining proceeds with a DP procedure that assigns a subscore and pointer to each cell, starting from the beginning of the cDNA sequence. By default, a mismatch within an exon incurs a penalty of two. Published by Oxford University Press. Further inspection of these 16 short intron/exon patterns and comparison of the alignments with available EST evidence suggests that these patterns may have been introduced computationally in order to maintain the reading frame. The genome may be read in as fasta files that contain either contigs in any order or entire assembled chromosomes. Other alignment programs had higher error rates than gmap on the unmutated data set. Strand selection in GeneSeqer depends on splice site scores, and the correct strand gives very poor splice sites. In other words, we implement the ssaha data structure for 12-mers, with the requirement that entries in the position table be pre-sorted in ascending numeric order within each oligomer. Distribution of oligomers of various lengths in the masked region of the human genome (NCBI build 29). For AY086065 and AY088578, the evidence appears to support the splice site in the gmap alignment, whereas for AY084877 and AY087013, the evidence appears to support the GeneSeqer splice site. Our implementation of 24-mer lookups on a genomic scale requires some adaptation of the index table scheme of ssaha (Ning et al., 2001). Another use is the construction of a genomic map file with gene boundaries (potentially overlapping). . In such cases, a program must decide whether extra nucleotides in the cDNA are due to a microexon or to an insertion in the adjoining exons. The middle graph shows the counts of ESTs whose genomic location by both programs overlap. In these examples, the predicted exons are indeed supported by other sequences, as listed in the figure. In experiment 1, we test for robustness to sequence error by using test sets of human mRNAs with computationally simulated sequence errors. Entries indicate the number of sequences with errors of various types. The top graph shows the total counts of ESTs at various quality levels. Probabilistic patterns of conservation are also seen at positions further away from the intronexon boundary (Mount, 1981; Senapathy et al., 1990; Solovyev, 2002). In all but 23 sequences (or 99.5% of the time), the two programs gave similar gene structures and splice sites. Programs can be overly liberal in identifying introns as being canonical, thereby resulting in false positives. Because the two programs report scores differently, we scored all alignments using the blast scoring system (Altschul et al., 1990), which assigns +1 point for matches, 3 for mismatches, 5 for gap openings and 2 for gap extensions, including the first nucleotide in the gap. If another valid mapping does not exist, the gene with lower identity is considered unmapped. One approach to this situation has been to combine information across various alignments (Birney et al., 2004; Haas et al., 2003; Brendel et al., 2004) or even multiple sources of evidence (Allen et al., 2004) to arrive at a consensus answer. In identifying gene structure, MGAlign came closest with 25 wrong sequences (2.8% error rate). The resulting cDNAgenomic alignments not only reveal the intronexon structure of genes, but also facilitate the study of splicing mechanics and such transcript-based phenomena as alternative splicing, single nucleotide polymorphisms, and cDNA insertions and deletions (Jiang and Jacob, 1998; Irizarry et al., 2000; Kan et al., 2001; Kan et al., 2002; Zavolan et al., 2002; Modrek and Lee, 2002; Clamp et al., 2003; Wheeler et al., 2003; Drabenstot et al., 2003; Kim et al., 2004; Florea et al., 2005). We thank William Wood and Scooter Morris for their support and encouragement. This is consistent with previous work showing the human genome and chimpanzee genome are approximately 98% identical (Chimpanzee Sequencing and Analysis Consortium, 2005). To handle this situation, after Liftoff maps all genes to their best matches, it checks for pairs of genes on the reference genome that have incorrectly mapped to overlapping (or identical) locations on the target genome, and it then attempts to find another valid mapping for one of the genes. For the first EST, which has one sequence difference relative to the genome, the canonical intron is recognized by five out of seven programs. For a single sequence, gmap is generally run in interactive mode, in which parts of the genomic files are read directly as needed. In this example, gmap and GeneSeqer are able to extend the alignment, thereby revealing a canonical intron and an additional exon. An optimal mapping is one in which the sequence identity is maximized while maintaining the integrity of each exon, transcript and gene. Existing cDNAgenomic mapping programs that use an oligomer index on a genomic scale begin by pre-loading the index into memory, which means that these programs not only have a long startup time, but also require computers with large amounts of dedicated RAM. In fact, 14-mers represent the current practical limit for the ssaha data structure, because the corresponding index file occupies 1.1 GB. This approach guarantees that all possible combinations of substitutions, gaps and intron shifts are considered, and permits the use of various DP techniques. Our minimal sampling strategy is based upon the use of long oligomers to achieve high specificity, combined with an adaptive sampling scheme to utilize mapping evidence from different parts of the cDNA sequence. The shortest path represents the combination of aligned blocks that is concordant with the original structure of the gene and minimizes the number of mismatches and indels within exons. Mapping and alignment of cDNA sequencesboth messenger RNAs (mRNAs) and expressed sequence tags (ESTs)onto the genome has become a central procedure in genome research. There are two types of genetic maps: 1) physical and 2) linkage. At run time, when a candidate genomic segment is found in the mapping step, gmap uses in subsequent alignment steps not only the genomic segment from the reference strain but also segments from relevant alternate strains by patching in the alternate strain sequence. Example of the lift-over process. However, when the alignment methodology was improved through oligomer chaining and sandwich DP, such matrices proved to be unnecessary, since the cDNA sequence (even with errors) plus the dinucleotide pairs at the end of the introns provide enough information to determine the splice site boundaries accurately. On the 1% data set, GeneSeqer had no gene structure errors and shifted canonical splice sites in 8 sequences. The functionality provided by gmap allows a user to: (1) map and align a single cDNA interactively against a large genome in about a second, without the startup time of several minutes typically needed by existing mapping programs; (2) switch arbitrarily among different genomes, without the need for a pre-loaded server dedicated to each genome; (3) run the program on computers with as little as 128 MB of RAM (random access memory); (4) perform high-throughput batch processing of cDNAs by using memory mapping and multithreading when appropriate memory and hardware are available; (5) generate accurate gene models, even in the presence of substantial polymorphisms and sequence errors; (6) locate splice sites accurately without the use of probabilistic splice site models, allowing generalized use of the program across species; (7) detect statistically significant microexons and incorporate them into the alignment; and (8) handle mapping and alignment tasks on genomes having alternate assemblies, linkage groups or strains. This sampling process is performed iteratively, with the sampling interval halved in each round. For full access to this pdf, sign in to an existing account, or purchase an annual subscription. Although minimal coverage of the genome can be achieved by sampling all non-overlapping 12-mers in the genome, an overlapping sampling interval provides increased resolution, but at the cost of a larger position file. GRCh37 and GRCh38 gene order. This article provides an overview of recent advancements in these fields, highlighting the role of bioinformatics in unraveling evolutionary insights and facilitating genome annotation. Alignments have been formatted in a uniform style. In comparison, gmap had 4 gene structure errors and 3 sequences with splicing errors. Genes that Liftoff failed to map are listed in Supplementary Table S3. The resulting margin gives the nucleotide-level DP procedure freedom to find a better alignment than that found by the coarser oligomer chaining procedure. In est_genome, approximate alignments are computed by using local Smith and Waterman (1981) alignments and the resulting segments are then recomputed with a global Needleman and Wunsch (1970) alignment. One approach to this problem is to try to improve the ability of the genomic mapping procedure to find the correct location initially. In addition, the co-linear mapping of genes from human chromosome 2 to chimpanzee chromosomes 2A and 2B is consistent with the known telomeric fusion of these chromosomes (Yunis et al., 1982). We classified errors into two classesgene structure errors and splicing errorsand counted the number of mRNAs for which an error occurred. (2003) in their simulation of observed errors in shotgun sequences. Sandwich alignments bridge large coordinate jumps across introns (horizontal dashed line) or long cDNA insertions (vertical dashed line). The optimal solution, shown in bold, is found by adding terms in adjacent rows, plus a reward for canonical introns, as indicated by the boxed GTAG pair. Compared with existing bisulfite mapping approaches, BSMAP is faster, more sensitive and more flexible. Like the Volfovsky method, our procedure searches for GT and AG pairs in the 5 and 3 ends surrounding the intron, but considers only those that satisfy the calculated lower bound on the microexon length. We used only the 4977 sequences for which the two programs agreed on gene structure. Genome mapping is used to identify and record the location of genes and the distances between genes on a chromosome. As we have mentioned, improvements in alignment accuracy can lead to improved genomic mapping of cDNAs, and hence result in better definitions of gene boundaries. 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