Aspergillus telomere to telomere genome assembly methods, apparatuses, devices, and storage media

Abstract

The application discloses an aspergillus telomere-to-telomere genome assembly method, device, equipment and storage medium. The method comprises the following steps: using a plurality of sequencing sequence assembly tools to assemble target aspergillus long read genome sequencing data from scratch to obtain a first assembled genome; selecting a first assembled genome meeting a preset condition as an initial assembled genome; integrating other first assembled genomes to fill gaps between repeat regions of the initial assembled genome to obtain a second assembled genome; aligning the long read genome sequencing data to the second assembled genome, identifying abnormal coverage regions and correcting sequences to obtain a third assembled genome; aligning a reference genome to the third assembled genome, connecting and orienting different contigs, and mounting the contigs to chromosomes to obtain a fourth assembled genome; and aligning the long read genome sequencing data and short read sequencing data to the fourth assembled genome for correction to obtain an aspergillus telomere-to-telomere genome assembly result.

Description

Technical Field

[0001] The embodiments of the present invention relate to bioinformatics processing technology, and more particularly to a method, apparatus, device and storage medium for assembling Aspergillus telomere to telomere genome. Background Technology

[0002] Aspergillus is a genus of filamentous fungi. From threatening human health as a pathogen to its applications in industrial fermentation and drug development, Aspergillus plays an irreplaceable role in many fields.

[0003] Aspergillus species have become a focus of research due to their significant medical and industrial value; however, the complexity of their genomes presents unique challenges for technological development. The rapid popularization of next-generation sequencing technologies (such as Illumina) has significantly reduced sequencing costs, enabling the completion of genome drafts for multiple Aspergillus species. However, the limitation of short read lengths (<300 bp) makes it difficult to traverse complex regions in the Aspergillus genome, such as the high proportion of repetitive sequences (e.g., transposable elements, rDNA clusters). These regions are crucial for chromosome stability and metabolic diversity, but their high homology leads to frequent breaks during the assembly of short-read data. For example, the first Aspergillus fumigatus genome sequence, Af293, is incomplete, containing deletions or gaps filled with unknown amino acids. Furthermore, the large evolutionary distances between Aspergillus species further increase the difficulty of cross-species comparisons and functional annotation.

[0004] In recent years, the groundbreaking development of third-generation sequencing technology has systematically solved the core bottlenecks of traditional sequencing technologies—the low throughput of Sanger sequencing and the short read lengths of second-generation sequencing—bringing a revolutionary change to the field of genome assembly. This has enabled telomere-to-telomere (T2T) genome assembly at the chromosome level to move from theory to practice. Third-generation technologies, represented by PacBio's continuous long read (CLR) mode (read length >15kb, accuracy 85%-97%) and high-fidelity (HiFi) mode (read length >10kb, error rate <0.5%), and Oxford Nanopore (ONT)'s standard (read length >15kb) and ultra-long (read length >50kb) sequencing methods (slightly lower in sequencing accuracy than HiFi reads), have achieved efficient traversal of complex and repetitive regions through the synergistic advantages of long read lengths and high accuracy. However, despite significant progress in genome research of some species, the usability of T2T assembly technology in Aspergillus genomes still faces serious challenges. Currently available Aspergillus genomes (such as Aspergillus fumigatus Af293) largely rely on next-generation sequencing data, resulting in highly fragmented assembly. Approximately 30%-40% of contigs cannot be anchored to chromosomes due to repetitive sequence folding or telomere loss. This contradiction highlights the urgent need to deeply integrate third-generation sequencing with T2T strategies into Aspergillus research to overcome the bottlenecks in repetitive sequence resolution and chromosome-level assembly, providing precise genetic maps for medical and industrial applications. Summary of the Invention

[0005] This invention provides a method, apparatus, device, and storage medium for assembling the Aspergillus telomere-to-telomere genome, so as to achieve complete and high-precision assembly of the Aspergillus telomere-to-telomere (T2T) genome.

[0006] In a first aspect, embodiments of the present invention provide a method for Aspergillus telomere-to-telomere genome assembly, comprising: Multiple sequencing assembly tools were used to de novo assemble the long-read genome sequencing data of the target Aspergillus, resulting in multiple first-assembled genomes. The first assembled genome that meets the preset conditions is selected based on preset indicators and used as the initial assembled genome; Using the initial assembled genome as a backbone, other first assembled genomes are integrated, and gaps are filled in the repetitive regions of the initial assembled genome to obtain a second assembled genome; The long-read genome sequencing data is aligned to the second assembled genome, regions with abnormal coverage are identified and sequence correction is performed to obtain the third assembled genome; The reference genome is aligned to the third assembled genome, different contigs are connected and oriented, and the contigs are mounted to chromosomes to obtain the fourth assembled genome; The long-read genome sequencing data and short-read sequencing data are aligned to the fourth assembled genome, and the fourth assembled genome is subjected to a preset round of base correction to obtain the genome assembly result of Aspergillus telomere to telomere.

[0007] Secondly, embodiments of the present invention also provide an Aspergillus telomere-to-telomere genome assembly apparatus, comprising: The first assembled genome determination module is used to de novo assemble the long-read genome sequencing data of the target Aspergillus using multiple sequencing sequence assembly tools to obtain multiple corresponding first assembled genomes; The initial assembled genome determination module is used to select the first assembled genome that meets the preset conditions based on preset indicators, as the initial assembled genome; The second assembled genome determination module is used to integrate other first assembled genomes with the initial assembled genome as a backbone, and to fill the gaps in the repetitive regions of the initial assembled genome to obtain the second assembled genome; The third assembled genome determination module is used to align the long-read genome sequencing data to the second assembled genome, identify regions with abnormal coverage and perform sequence correction to obtain the third assembled genome. The fourth assembled genome determination module is used to align the reference genome to the third assembled genome, connect and orient different contigs, and attach the contigs to chromosomes to obtain the fourth assembled genome; The genome assembly result determination module is used to align the long-read genome sequencing data and short-read sequencing data to the fourth assembled genome, perform a preset round of base correction on the fourth assembled genome, and obtain the genome assembly result of Aspergillus telomere to telomere.

[0008] Thirdly, embodiments of the present invention also provide a computer device, comprising: One or more processors; Memory, used to store one or more programs; When the one or more programs are executed by the one or more processors, the one or more processors implement the Aspergillus telomere-to-telomere genome assembly method provided in any embodiment of the present invention.

[0009] Fourthly, embodiments of the present invention also provide a storage medium containing computer-executable instructions, which, when executed by a computer processor, are used to perform the Aspergillus telomere-to-telomere genome assembly method provided in any embodiment of the present invention.

[0010] This invention constructs a continuous assembly backbone using long-read genome sequencing data. Then, through cross-validation and intelligent decision-making based on multi-source assembly results, it precisely fills complex gaps in the assembly backbone and completes telomere sequences. Combined with short-read data, the genome is corrected to ensure ultra-high accuracy at the base level. This addresses the problem that assembly strategies based primarily on short-read sequencing data cannot cross long repetitive sequences, resulting in limited continuous sequence length, while most Aspergillus genome sequences still contain many gaps. This invention achieves a complete and accurate Aspergillus T2T genome. Attached Figure Description

[0011] Figure 1 This is a flowchart of a method for assembling Aspergillus telomeres to telomeres genome according to an embodiment of the present invention; Figure 2 This is a flowchart of the Aspergillus T2T genome assembly process in an embodiment of the present invention; Figure 3 This is a schematic diagram of the structure of an Aspergillus telomere-to-telomere genome assembly device according to an embodiment of the present invention; Figure 4 This is a schematic diagram of the structure of a computer device according to an embodiment of the present invention. Detailed Implementation

[0012] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, the accompanying drawings show only the parts relevant to the present invention, and not all of the structures.

[0013] Figure 1 This is a flowchart of the Aspergillus telomere-to-telomere genome assembly method provided in an embodiment of the present invention. This embodiment is applicable to the case of Aspergillus T2T genome assembly. The method can be executed by an Aspergillus telomere-to-telomere genome assembly device, which can be implemented by hardware and / or software and is generally integrated into an electronic device, such as a computer device. The method specifically includes the following steps: Step 110: Use multiple sequencing assembly tools to de novo assemble the long-read genome sequencing data of the target Aspergillus, and obtain multiple corresponding first-assembled genomes.

[0014] The target *Aspergillus* strain is the *Aspergillus* strain requiring genome assembly. The *Aspergillus* genome is pre-sequentially sequenced using long-read sequencing to obtain long-read genome sequencing data, or this data can be obtained from existing databases. For example, PacBio's high-fidelity (HiFi) sequencing technology can be used to obtain the full-length genome data of the target *Aspergillus* strain. Furthermore, rigorous length screening and quality control measures, such as removing reads shorter than 500 bp and extracting high-abundance reads (HIFI), are applied to the raw reads before assembly to obtain raw data that possesses both long read length and high base accuracy. Multiple sequencing sequence assembly tools are used to assemble the long-read genome data de novo. Each sequencing sequence assembly tool assembles a first-assembled genome, meaning there is a one-to-one correspondence between the first-assembled genome and the sequencing sequence assembly tool. This embodiment of the invention introduces a multi-tool competitive evaluation to ensure genome continuity and effectively avoid the systematic biases that may exist in a single assembly algorithm, thus facilitating the subsequent selection of the optimal backbone.

[0015] Optionally, multiple sequencing assembly tools are used to de novo assemble the long-read genome sequencing data of the target Aspergillus, resulting in multiple corresponding first-assembled genomes, including: Multiple sequencing sequence assembly tools are selected and default parameters are set to assemble the long-read genome test data de novo, resulting in a first assembled genome corresponding to each sequencing sequence assembly tool; wherein, the sequencing sequence assembly tools include at least two of Flye, Hifiasm, and Hicanu. For example, the genome de novo assembly tools Flye (v2.8.2), Hifiasm (v0.16.1), and Hicanu (v2.2) for third-generation sequencing data are used to independently assemble the same set of high-quality HiFi sequencing data de novo (with default parameters set), resulting in three first assembled genomes.

[0016] Step 120: Select the first assembled genome that meets the preset conditions based on preset indicators, and use it as the initial assembled genome.

[0017] This process involves comparing multiple first-assembled genomes based on preset indicators, selecting those that meet pre-defined criteria. These indicators may include continuity and integrity metrics. By systematically comparing the differences among first-assembled genomes in key indicators such as continuity (e.g., N50) and integrity (e.g., number of contigs), the first-assembled genome with the best global continuity performance is intelligently selected as the initial assembled genome. Multiple threshold intervals can be set to evaluate continuity and integrity. The corresponding indicators of the first-assembled genome are matched with these threshold intervals to obtain corresponding scores. The scores of both continuity and integrity are then combined to obtain the final score for the first-assembled genome. The scores of multiple first-assembled genomes are then compared to select the optimal one as the initial assembled genome. For example, the final comparison shows that the Flye assembly not only covers a longer genomic region but also yields the fewest initial contigs, making it a suitable initial assembled genome.

[0018] Optionally, the first assembled genome that meets preset conditions is selected as the initial assembled genome based on a continuity index, including: Compare multiple first-assembled genomes using pre-defined metrics; these metrics include continuity metrics and integrity metrics. Based on the comparison results of preset indicators, the optimal first assembled genome is selected as the initial assembled genome.

[0019] Step 130: Using the initial assembled genome as a backbone, integrate other first assembled genomes, fill the gaps in the repetitive regions of the initial assembled genome, and obtain the second assembled genome.

[0020] The initially assembled genome typically contains gaps in repetitive regions, primarily caused by highly repetitive sequences such as centromeres and rDNA. To improve the integrity of the assembly results, this invention proposes a gap-filling model for cross-validation and intelligent decision-making in multi-source assembly. This model uses the initially assembled genome as a backbone, which is denoted as a sequence set. k is the total number of skeleton sequences, which contain a number of gaps ( m is the total number of gaps, and each gap In one of the sequences Above, and with "left anchor sequences" and "right anchor sequences" in between, the first assembled genome assembled independently is regarded as a dynamic "sequence patch library" (with the above, and with "left anchor sequences" and "right anchor sequences" in between). t represents the number of assembly results in the sequence patch library, each It is a set of one or more assembly result sequences.

[0021] For each gap ∈G, find the left anchoring region and the right anchorage area (For example, the anchoring length 'a' can be 100bp, 200bp, 300bp up to 1000bp):

[0022] Where p and q are the gaps, respectively. The left and right boundary positions on the skeleton sequence. Assemble each source from the sequence patch library P. Find all sequence segments c∈ It satisfies the requirement that there is a certain overlap or alignment with the left and right anchoring areas, i.e., the overlap length threshold. The set of candidate sequence segments for the j-th gap is:

[0023] Or at least one side overlaps, that is, one end is aligned and the other end falls within the gap area. For sequence segment c and the left anchoring region The overlap length, For sequence segment c and the right anchoring region The overlap length. For each read in the candidate set that falls within the gap expansion region, take the corresponding subsequence from the substring (or the entire read) of that read, perform k-mer splitting, and for each such read... The k-mer set of a single read segment r ,make

[0024] The set of all these k-mers is denoted as Let t be the starting position of the k-mer in the read segment r, and then expand from both the left and right sides to close the gap. The left anchor extension is a series of k-mers extending from the left boundary into the gap, denoted as the left extension set. The series of k-mers extending from the right boundary into the gap at the right anchor end are denoted as... These extended k-mers require an overlap between adjacent k-mers: if The last k-1 nucleotides of one k-mer match the first k-1 nucleotides of another k-mer, allowing for splicing; the same applies to the right side. Let the overlap function be:

[0025] Among them, suffix k-1 (k a ) refers to k a The last k-1 bases, prefix k-1 (kb ):k b The first k-1 bases. k a and k b It is any two k-mers in the k-mer set.

[0026] If a path or chain exists , The starting k-mer of the left-hand extended set, The terminating k-mer of the right-hand extended set, For connection and k-mer paths, where adjacent k-mers satisfy for all have

[0027] Furthermore, the length of the entire merged sequence is approximately equal to the estimated gap length. (Tolerable error threshold) ):

[0028] Let be the concatenation function for k-mer paths, where each k-mer in the path originates from reads ≥ 1. , If the threshold for the number of read segments supported by k-mer is used, then the gap is considered to be... It is closed, creating a gap. The optimal imputation sequence:

[0029] This enables highly reliable filling of complex regions such as centromeres. Combining the advantages of the assembly results from the three tools, a highly continuous and accurate second-assembled genome is ultimately obtained.

[0030] Step 140: Align the long-read genome sequencing data to the second assembled genome, identify regions with abnormal coverage and perform sequence correction to obtain the third assembled genome.

[0031] This process involves mapping raw PacBio HiFi long-read genome sequencing data to a second assembled genome for data consistency-based assembly quality assessment. By analyzing genome-wide read coverage, regions with abnormal coverage are systematically identified, often indicating potential assembly errors. These abnormal regions are then subject to focused review and local optimization to correct potential chimeric connections or repetitive sequence collapse, resulting in a third assembled genome with improved global consistency and local accuracy.

[0032] Step 150: Align the reference genome to the third assembled genome, connect and orient different contigs, attach the contigs to chromosomes, and obtain the fourth assembled genome.

[0033] In order to effectively correct potential problems in the assembly process and ensure the accuracy and integrity of the genome assembly, MUMmer (e.g., version 4.0.0) can be used to compare the third assembled genome with the reference genome in the NCBI RefSeq database. Based on the comparison results, different contigs are connected and oriented, and then mounted onto the corresponding chromosomes to obtain the fourth assembled genome.

[0034] Step 160: Align the long-read genome sequencing data and short-read sequencing data to the fourth assembled genome, perform a preset round of base correction on the fourth assembled genome, and obtain the genome assembly results of Aspergillus telomeres to telomeres.

[0035] The *Aspergillus* genome was sequenced using the Illumina high-throughput sequencing platform, yielding short reads from both ends. For the raw Illumina FastQ sequencing data, the FastP software was used for rigorous quality control, removing reads with more than 15 bp overlap with adapter sequences and fewer than 3 mismatches, N base content exceeding the 10% threshold, and low-quality bases (quality value ≤20) comprising more than 40% of the reads (default parameters), resulting in clean reads with a high-precision calibration baseline. To improve assembly accuracy, a dual-round iterative calibration strategy leveraging the complementary advantages of heterogeneous data was employed for the fourth-assembled genome, ultimately yielding the *Aspergillus* T2T genome assembly results. The first round of calibration utilized the long read length and high precision of the PacBio HiFi data, using Racon (v1.6.0) for global, large-scale consensus optimization of the fourth-assembled genome, focusing on correcting base errors and local mismatches. The second round of correction utilizes the extremely high single-base precision of Illumina data, using Pilon (v1.24) for fine correction (parameters –changes –vcf) to eliminate residual single nucleotide errors and small insertions / deletions (Indels), ultimately improving the overall base quality of the genome to the T2T standard requirements.

[0036] This invention employs a competitive evaluation strategy for multi-tool genome assembly results, utilizing a gap-filling model combining cross-validation of multi-source genome assembly results and intelligent decision-making to effectively compensate for the shortcomings of single tools in handling repetitive or low-coverage regions. Through this complementary strategy, the overall assembly quality is significantly improved, increasing both the Contig N50 value and the integrity and accuracy of the assembly results. Secondly, the multi-tool combination scheme reduces the error rate caused by the limitations of a single algorithm. Mutual validation between different algorithms effectively corrects potential assembly errors and redundant sequences, resulting in a genome that more closely approximates reality. Furthermore, this invention fully leverages the advantages of third-generation sequencing data—both its long read length and high accuracy—providing an efficient and reliable solution for Aspergillus genome research.

[0037] Optionally, using the initial assembled genome as a backbone, other first-assembled genomes are integrated, and gaps are filled in the repetitive regions of the initial assembled genome to obtain a second-assembled genome, including: Using the initial assembled genome as a backbone, flanking sequences between each repetitive region in the initial assembled genome were extracted as anchoring sequences; Using the assembly sequences of other first-assembled genomes as reference sequences, the anchor sequence is compared with the reference sequence to search for regions in the reference sequence that match the anchor sequence; If a pair of anchor sequences finds a matching region in the same reference sequence, the sequence from the reference sequence is extracted to replace the corresponding gap in the initially assembled genome, thereby filling the gaps in the repetitive regions remaining in the initially assembled genome.

[0038] Optionally, after chromosome mounting is completed, a telomere sequence scanning and verification algorithm specifically designed for chromosome ends is used. After aligning the reference genome to the third assembled genome, connecting and orienting different contigs, mounting the contigs to chromosomes, and obtaining the fourth assembled genome, the process also includes: The telomere sequences designed for chromosome ends were scanned and verified; where the total number of chromosomes is C, the end sequences at the left and right ends of each chromosome c are defined as follows:

[0039] in, This is the left end sequence of the chromosome. This is the right-end sequence of the chromosome. This is the base sequence of chromosome c. The length of chromosome c. The end proportion parameter (default d=0.01, i.e., 1% at each end) and all the following statistics are based on the end sequence set of all chromosomes. Cumulative calculation; For a candidate motif, the length k of the motif, The minimum length of the candidate motif. Define the maximum length of the candidate motif, define the serial repetition statistics at its ends, and predefine the sequence at the ends. At position i, define the maximum number of consecutive series connections for candidate modulus m:

[0040] in This indicates r consecutive repetitions of m; if there is no match, then... The cumulative statistic is defined as (summing over all ends):

[0041]

[0042]

[0043] in, This indicates the number of repetition start points, where m is the number of times the serial repetition start point appears; This represents the number of duplicate copies, where m is the total number of repeating units (including partial overlap). This indicates the number of repeating bases, representing the total base length occupied by all repeating units.

[0044] To measure end enrichment, a baseline background statistic (in non-end regions or other locations throughout the genome) is introduced: the total number of bases covered by motif m in the background region. With corresponding background length Define the coverage ratio of the end series connection:

[0045] Background coverage ratio:

[0046] Define enrichment fold change:

[0047] This avoids introducing a small constant ε during division by zero; To describe the "distribution rationality" (end clustering / serialization characteristics), the ends are divided into B bins, and the initial number of each bin is denoted as . probability distribution Location clustering score definition:

[0048] in, Shannon entropy of the motif measures the degree of dispersion of the motif at its ends. The smaller the value (closer to 0), the more concentrated the motif's initiation position is in a few end regions, which conforms to the aggregation characteristics of telomere sequences. This represents the location clustering score, measuring whether the area is concentrated in a few regions (the closer to 1, the more clustered). The overall score S(m) is defined as follows:

[0049] Each sub-fraction is first normalized to [0,1]. , , , The weighting coefficients for each sub-score, each weighting coefficient 0, , , , These are the end coverage, enrichment, and maximum concatenation length after normalization to the maximum or logarithmic scale, respectively; finally, the candidate selection and judgment rules (hard threshold + score) are given:

[0050] satisfy , , ,but True. And the final telomere repeat sequence is selected as:

[0051] This algorithm identifies chromosomes with intact telomeres as well as chromosome ends with missing or incomplete telomeres. It not only highly sensitively identifies classic telomere repeat units and their variants but also effectively handles natural variations in the number of repeat units. More importantly, the process can accurately determine the position and orientation of telomere sequences on chromosomes, thus clearly identifying chromosomes with intact telomeres as well as chromosome ends with missing or incomplete telomeres.

[0052] Optionally, long-read and short-read genome sequencing data are aligned to the fourth-order genome assembly, and the fourth-order genome assembly undergoes a preset round of base correction to obtain the Aspergillus telomere-to-telomere genome assembly results, including: Based on long-read genome sequencing data, Racon was used to perform the first round of global correction on the fourth assembled genome, correcting base errors and local mismatches. Based on short-read sequencing data, a second round of correction was performed using Pilon to eliminate remaining single-base errors and small insertions / deletions.

[0053] Optionally, after aligning the long-read genome sequencing data and short-read sequencing data to the fourth-assembled genome, performing a preset round of base correction on the fourth-assembled genome, and obtaining the Aspergillus telomere-to-telomere genome assembly results, the process further includes: The assembly size of the genome assembly results, the sum of all allele lengths, and N50 were calculated using QUAST. The integrity of the genome assembly results was evaluated by comparing the fungal core gene set with BUSCO; BUSCO version v5.2.2 can be used, and the fungal core gene set can be FUNGI_ODB10.

[0054] Based on long-read and short-read genome sequencing data, a hybrid k-mer database was constructed using Merqury to assess the base identity between the assembled sequences and the original reads. For example, based on Illumina short reads and PacBio HiFi long reads, a hybrid k-mer database (k-mer=17bp) was constructed using Merqury (v1.3) to assess the base identity between the assembled sequences and the original reads (quality value QV not less than 50).

[0055] After obtaining the Aspergillus T2T genome assembly results, the results can be evaluated in terms of three dimensions: continuity, accuracy, and completeness.

[0056] In a preferred embodiment, such as Figure 2 The flowchart shown illustrates the assembly process of the Aspergillus T2T genome. The full-sequence data of the target Aspergillus genome was obtained using PacBio's high-fidelity (HiFi) sequencing technology. Three assembly tools—Flye, Hifiasm, and Hicanu—were used to independently assemble the same set of PacBio sequencing data from scratch. By comparing the differences in continuity and completeness among the assembled genomes, the assembly version with the best global continuity was selected as the initial genome. Gap filling was performed on the initial genome to obtain a second assembled genome with high continuity and accuracy. The original PacBio long-read sequencing data was mapped to the second assembled genome for assembly error correction, resulting in the third assembled genome. The third assembled genome was aligned with a reference genome in the NCBI RefSeq database. Based on the alignment results, different contigs were joined and oriented, and then mounted onto the corresponding chromosomes to obtain the fourth assembled genome. The first round of correction was performed using Racon with PacBio sequencing data, and the second round of correction was performed using Pilon with Illumina sequencing data, ultimately assembling the T2T genome.

[0057] In an exemplary embodiment, based on the above main steps and combining PacBio high-fidelity (HiFi) long-read sequencing data (average coverage depth 116.9×) and Illumina short-read sequencing data (average coverage depth 88.9×), this embodiment of the invention successfully achieved chromosome-level assembly of the genomes of 26 Aspergillus strains. Seven Aspergillus fumigatus strains had genome sizes of 28.5-29.4 Mb, N50 values ​​of 3.8-4.1 Mb, and 9-10 contigs, all corresponding to 8 chromosomes. Five of these strains contained only 9 contigs, indicating that each chromosome was covered by a single contig, with only one contig unanchored, achieving near-chromosome-level assembly continuity. The remaining two strains contained 10 contigs, with two unlocalized segments, but still superior to traditional assembly. The Aspergillus flavus genome size was 37.6-38.2 Mb (largest among Aspergillus strains), N50 values ​​of 4.2-4.7 Mb, and 9-11 contigs. Of the 11 contigs in the strains, 3 contigs were not anchored to all 8 chromosomes, but their N50 values ​​were still significantly better than those of next-generation sequencing. Four *Aspergillus niger* strains had genome sizes of 36.2–37.4 Mb and 9–10 contigs. Of the 10 contigs in the strains, 2 were not anchored, but their N50 values ​​(4.2–4.9 Mb) indicated complete coverage of major chromosomal regions. *Aspergillus japonicus*, with the largest genome, had the highest N50 value (6.1 Mb), containing only 9 contigs corresponding to 8 chromosomes, with 1 unlocated contig, indicating efficient traversal of its repetitive sequences. *Aspergillus montana*, with the smallest genome and lowest N50 value, covered 8 chromosomes with 9 contigs, demonstrating stable continuity. The number of contigs in all species was close to the number of chromosomes, and the N50 value of the contigs was significantly higher than or comparable to the T2T level genomes of previously sequenced *Aspergillus* species (Table 1, statistics of *Aspergillus* T2T genome assembly results). Simultaneously, this invention successfully reconstructed telomeres at one or both ends of the chromosome for 26 strains, of which 15 strains achieved complete assembly without missing telomeres or gaps (Table 2, statistics of Aspergillus gap locations and telomere deletion locations). In all strains, over 98.5% of Illumina short reads and 99.5% of PACBIO HIFI reads were successfully mapped back to the final assembly results, indicating high accuracy. Furthermore, BUSCO evaluation revealed that, on average, 98.7% of single-copy fungal genes (FUNGI_ODB10) were completely assembled in these genomes. High BUSCO scores and homolinearity with the NCBI reference genome demonstrate the quality and completeness of our assembly (Table 3, evaluation of Aspergillus T2T genome assembly results).

[0058] Table 1

[0059]

[0060] Table 2

[0061] Table 3

[0062]

[0063]

[0064] like Figure 3 As shown, this embodiment of the invention also provides an Aspergillus telomere-to-telomere genome assembly device, comprising: The first assembled genome determination module 310 is used to de novo assemble the long-read genome sequencing data of the target Aspergillus using multiple sequencing sequence assembly tools to obtain multiple corresponding first assembled genomes. The initial assembled genome determination module 320 is used to select the first assembled genome that meets the preset conditions based on preset indicators as the initial assembled genome; The second assembled genome determination module 330 is used to integrate other first assembled genomes with the initial assembled genome as a backbone, and to fill the gaps in the repetitive regions of the initial assembled genome to obtain the second assembled genome; The third assembled genome determination module 340 is used to align the long-read genome sequencing data to the second assembled genome, identify regions with abnormal coverage and perform sequence correction to obtain the third assembled genome. The fourth assembled genome determination module 350 is used to align the reference genome to the third assembled genome, connect and orient different contigs, and attach the contigs to chromosomes to obtain the fourth assembled genome. The genome assembly result determination module 360 ​​is used to align the long-read genome sequencing data and short-read sequencing data to the fourth assembled genome, perform a preset round of base correction on the fourth assembled genome, and obtain the genome assembly result of Aspergillus telomere to telomere.

[0065] Optionally, the first assembly genome determination module is specifically used for: Multiple sequencing sequence assembly tools are selected and default parameters are set to assemble the long-read genome test data de novo, resulting in the first assembled genome corresponding to each sequencing sequence assembly tool; wherein, the sequencing sequence assembly tools include at least two of Flye, Hifiasm, and Hicanu.

[0066] Optional, an initial assembly genome determination module, specifically used for: Compare multiple first-assembled genomes using pre-defined metrics; these metrics include continuity metrics and integrity metrics. Based on the comparison results of preset indicators, the optimal first assembled genome is selected as the initial assembled genome.

[0067] Optionally, a second assembly genome determination module is used for: Using the initial assembled genome as a backbone, flanking sequences between each repetitive region in the initial assembled genome were extracted as anchoring sequences; Using the assembly sequences of other first-assembled genomes as reference sequences, the anchor sequence is compared with the reference sequence to search for regions in the reference sequence that match the anchor sequence; If a pair of anchor sequences finds a matching region in the same reference sequence, the sequence from the reference sequence is extracted to replace the corresponding gap in the initially assembled genome, thereby filling the gaps in the repetitive regions remaining in the initially assembled genome.

[0068] Optionally, the Aspergillus telomere-to-telomere genome assembly device also includes: The telomere sequence scanning and verification module is used to scan and verify telomere sequences designed for chromosome ends after aligning the reference genome to the third assembled genome, connecting and orienting different contigs, attaching the contigs to chromosomes, and obtaining the fourth assembled genome. The total number of chromosomes is C, and the end sequences at the left and right ends of each chromosome c are defined as follows:

[0069] in This is the base sequence of chromosome c. The length of chromosome c. The end-scale parameter is a parameter and all the following statistics are in the set. Cumulative calculation; For candidate phantoms, the length k of the phantom, Define its serial repetition statistics at the ends, with a preset sequence at the ends. Define the maximum number of consecutive series at position i:

[0070] in This indicates r consecutive repetitions of m; if there is no match, then... The cumulative statistic is defined as:

[0071]

[0072]

[0073] To measure end-point enrichment, baseline background statistics are introduced. With corresponding background length Define the end concatenation ratio and background coverage ratio:

[0074] Define enrichment factor:

[0075] This avoids introducing a small constant ε during division by zero; Divide the end into B bins, and denote the initial number of each bin as . probability distribution Location clustering score definition:

[0076] in A value approaching 0 indicates that the distribution is concentrated at the terminal poles, consistent with telomere characteristics. The comprehensive score S(m) is defined as follows:

[0077] Each sub-fraction is first normalized to [0,1]. 0, , , , These are the end coverage, enrichment, and maximum concatenation length after normalization to the maximum or logarithmic scale, respectively; finally, the candidate selection and judgment rules are given:

[0078] And the final telomere repeat sequence was selected as:

[0079] To identify chromosomes with intact telomeres and chromosome ends with missing or incomplete telomeres.

[0080] Optional, a genome assembly result determination module, specifically used for: Based on long-read genome sequencing data, Racon was used to perform the first round of global correction on the fourth assembled genome, correcting base errors and local mismatches. Based on short-read sequencing data, a second round of correction was performed using Pilon to eliminate remaining single-base errors and small insertions / deletions.

[0081] Optionally, the Aspergillus telomere-to-telomere genome assembly device also includes: The assembly result quality assessment module is used to compare long-read genome sequencing data and short-read sequencing data to the fourth assembled genome, perform a preset round of base correction on the fourth assembled genome, and obtain the Aspergillus telomere-telomere genome assembly result. Then, it uses QUAST to calculate the assembly size of the genome assembly result, the sum of the lengths of all alleles, and N50. The integrity of the genome assembly results was assessed using the BUSCO alignment of the fungal core gene set; Based on long-read genome sequencing data and short-read sequencing data, a hybrid k-mer database was constructed using Merqury to assess the base identity between the assembled sequence and the original reads.

[0082] The above-mentioned products can perform the Aspergillus telomere-to-telomere genome assembly method provided in any embodiment of the present invention, and have the corresponding functional modules and beneficial effects of performing the method.

[0083] Figure 4 A schematic diagram of the structure of a computer device provided in an embodiment of the present invention, such as... Figure 4 As shown, the computer device includes a processor 410, a memory 420, an input device 430, and an output device 440; the number of processors 410 in the computer device can be one or more. Figure 4 Taking a processor 410 as an example; the processor 410, memory 420, input device 430, and output device 440 in a computer device can be connected via a bus or other means. Figure 4 Taking the example of a connection between China and Israel via a bus.

[0084] The memory 420, as a computer-readable storage medium, can be used to store software programs, computer-executable programs, and modules, such as the program instructions / modules corresponding to the Aspergillus telomere-to-telomere genome assembly method in this embodiment of the invention (e.g., the first assembled genome determination module 310, the initial assembled genome determination module 320, the second assembled genome determination module 330, the third assembled genome determination module 340, the fourth assembled genome determination module 350, and the genome assembly result determination module 360 ​​in the Aspergillus telomere-to-telomere genome assembly device). The processor 410 executes various functional applications and data processing of the computer device by running the software programs, instructions, and modules stored in the memory 420, thereby realizing the above-described Aspergillus telomere-to-telomere genome assembly method.

[0085] The memory 420 may primarily include a program storage area and a data storage area. The program storage area may store the operating system and at least one application program required for a given function; the data storage area may store data created based on terminal usage. Furthermore, the memory 420 may include high-speed random access memory and non-volatile memory, such as at least one disk storage device, flash memory, or other non-volatile solid-state storage device. In some instances, the memory 420 may further include memory remotely located relative to the processor 410, which can be connected to the computer device via a network. Examples of such networks include, but are not limited to, the Internet, intranets, local area networks, mobile communication networks, and combinations thereof.

[0086] Input device 430 can be used to receive input digital or character information, and to generate key signal inputs related to user settings and function control of the computer device. Output device 440 may include display devices such as a display screen.

[0087] This invention also provides a storage medium containing computer-executable instructions, which, when executed by a computer processor, are used to perform an Aspergillus telomere-to-telomere genome assembly method, including: Multiple sequencing assembly tools were used to de novo assemble the long-read genome sequencing data of the target Aspergillus, resulting in multiple first-assembled genomes. The first assembled genome that meets the preset conditions is selected based on preset indicators and used as the initial assembled genome; Using the initial assembled genome as a backbone, other first assembled genomes are integrated, and gaps are filled in the repetitive regions of the initial assembled genome to obtain a second assembled genome; The long-read genome sequencing data is aligned to the second assembled genome, regions with abnormal coverage are identified and sequence correction is performed to obtain the third assembled genome; The reference genome is aligned to the third assembled genome, different contigs are connected and oriented, and the contigs are mounted to chromosomes to obtain the fourth assembled genome; The long-read genome sequencing data and short-read sequencing data are aligned to the fourth assembled genome, and the fourth assembled genome is subjected to a preset round of base correction to obtain the genome assembly result of Aspergillus telomere to telomere.

[0088] Of course, the computer-executable instructions provided in the embodiments of the present invention are not limited to the method operations described above, but can also perform related operations in the Aspergillus telomere-to-telomere genome assembly method provided in any embodiment of the present invention.

[0089] Based on the above description of the implementation methods, those skilled in the art can clearly understand that the present invention can be implemented using software and necessary general-purpose hardware, and of course, it can also be implemented using hardware, but in many cases the former is a better implementation method. Based on this understanding, the technical solution of the present invention, or the part that contributes to the prior art, can be embodied in the form of a software product. This computer software product can be stored in a computer-readable storage medium, such as a computer floppy disk, read-only memory (ROM), random access memory (RAM), flash memory, hard disk, or optical disk, etc., including several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute the methods described in the various embodiments of the present invention.

[0090] It is worth noting that in the above embodiments of the Aspergillus telomere-to-telomere genome assembly device, the various units and modules included are only divided according to functional logic, but are not limited to the above division, as long as the corresponding functions can be achieved; in addition, the specific names of each functional unit are only for easy differentiation and are not used to limit the scope of protection of the present invention.

[0091] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for assembling Aspergillus telomeres to telomere genomes, characterized in that, include: Multiple sequencing assembly tools were used to de novo assemble the long-read genome sequencing data of the target Aspergillus, resulting in multiple first-assembled genomes. The first assembled genome that meets the preset conditions is selected based on preset indicators and used as the initial assembled genome; Using the initial assembled genome as a backbone, other first assembled genomes are integrated, and gaps are filled in the repetitive regions of the initial assembled genome to obtain a second assembled genome; The long-read genome sequencing data is aligned to the second assembled genome, regions with abnormal coverage are identified and sequence correction is performed to obtain the third assembled genome; The reference genome is aligned to the third assembled genome, different contigs are connected and oriented, and the contigs are mounted to chromosomes to obtain the fourth assembled genome; The long-read genome sequencing data and short-read sequencing data are aligned to the fourth assembled genome, and the fourth assembled genome is subjected to a preset round of base correction to obtain the genome assembly result of Aspergillus telomere to telomere.

2. The method according to claim 1, characterized in that, Multiple sequencing assembly tools were used to de novo assemble the long-read genome sequencing data of the target Aspergillus, resulting in multiple corresponding first-assembled genomes, including: Multiple sequencing sequence assembly tools are selected and default parameters are set to assemble the long-read genome test data de novo, resulting in the first assembled genome corresponding to each sequencing sequence assembly tool; wherein, the sequencing sequence assembly tools include at least two of Flye, Hifiasm, and Hicanu.

3. The method according to claim 1, characterized in that, The first assembled genome that meets preset conditions based on continuity indicators is selected as the initial assembled genome, including: Compare the preset indicators of multiple first assembled genomes; wherein the preset indicators include continuity indicators and integrity indicators; Based on the comparison results of preset indicators, the optimal first assembled genome is selected as the initial assembled genome.

4. The method according to claim 1, characterized in that, Using the initial assembled genome as a backbone, other first assembled genomes are integrated, and gaps are filled in the repetitive regions of the initial assembled genome to obtain a second assembled genome, comprising: Using the initial assembled genome as a backbone, flanking sequences of each repetitive region gap in the initial assembled genome are extracted as anchoring sequences; Using other assembled sequences of the first assembled genome as reference sequences, the anchor sequence is compared with the reference sequence to search for regions in the reference sequence that match the anchor sequence; If a pair of anchor sequences finds a matching region in the same reference sequence, the sequence of the reference sequence is extracted to replace the corresponding gap in the initially assembled genome to fill the gaps in the repetitive regions remaining in the initially assembled genome.

5. The method according to claim 1, characterized in that, After aligning the reference genome to the third assembled genome, connecting and orienting different contigs, and attaching the contigs to chromosomes to obtain the fourth assembled genome, the process further includes: The telomere sequences designed for chromosome ends were scanned and verified; where the total number of chromosomes is C, the end sequences at the left and right ends of each chromosome c are defined as follows: in This is the base sequence of chromosome c. The length of chromosome c. The end-scale parameter is a parameter and all the following statistics are in the set. Cumulative calculation; For candidate phantoms, the length k of the phantom, Define its serial repetition statistics at the ends, with a preset sequence at the ends. Define the maximum number of consecutive series at position i: in This indicates r consecutive repetitions of m; if there is no match, then... The cumulative statistic is defined as: To measure end-point enrichment, baseline background statistics are introduced. With corresponding background length Define the end concatenation ratio and background coverage ratio: Define enrichment factor: This avoids introducing a small constant ε during division by zero; Divide the end into B bins, and denote the initial number of each bin as . probability distribution Location clustering score definition: in A value approaching 0 indicates that the distribution is concentrated at the terminal poles, consistent with telomere characteristics. The comprehensive score S(m) is defined as follows: Each sub-fraction is first normalized to [0,1]. 0, , , , These are the end coverage, enrichment, and maximum concatenation length after normalization to the maximum or logarithmic scale, respectively; finally, the candidate selection and judgment rules are given: And the final telomere repeat sequence was selected as: To identify chromosomes with intact telomeres and chromosome ends with missing or incomplete telomeres.

6. The method according to claim 1, characterized in that, The long-read genome sequencing data and short-read sequencing data are aligned to the fourth assembled genome, and the fourth assembled genome undergoes a preset round of base correction to obtain the Aspergillus telomere-to-telomere genome assembly result, including: Based on the long-read genome sequencing data, Racon was used to perform the first round of global correction on the fourth assembled genome to correct base errors and local mismatches. Based on the short read sequencing data, a second round of correction was performed using Pilon to eliminate remaining single-base errors and small insertions / deletions.

7. The method according to claim 6, characterized in that, After aligning the long-read genome sequencing data and short-read sequencing data to the fourth assembled genome, performing a preset round of base correction on the fourth assembled genome to obtain the Aspergillus telomere-to-telomere genome assembly result, the process further includes: The assembly size of the genome assembly result, the sum of all allele lengths, and N50 were calculated using QUAST. The integrity of the genome assembly results was assessed using the BUSCO alignment of the fungal core gene set; Based on the long-read genome sequencing data and the short-read sequencing data, a hybrid k-mer database was constructed using Merqury to evaluate the base identity between the assembled sequence and the original reads.

8. An Aspergillus telomere-to-telomere genome assembly device, characterized in that, include: The first assembled genome determination module is used to de novo assemble the long-read genome sequencing data of the target Aspergillus using multiple sequencing sequence assembly tools to obtain multiple corresponding first assembled genomes; The initial assembled genome determination module is used to select the first assembled genome that meets the preset conditions based on preset indicators, as the initial assembled genome; The second assembled genome determination module is used to integrate other first assembled genomes with the initial assembled genome as a backbone, and to fill the gaps in the repetitive regions of the initial assembled genome to obtain the second assembled genome; The third assembled genome determination module is used to align the long-read genome sequencing data to the second assembled genome, identify regions with abnormal coverage and perform sequence correction to obtain the third assembled genome. The fourth assembled genome determination module is used to align the reference genome to the third assembled genome, connect and orient different contigs, and attach the contigs to chromosomes to obtain the fourth assembled genome; The genome assembly result determination module is used to align the long-read genome sequencing data and short-read sequencing data to the fourth assembled genome, perform a preset round of base correction on the fourth assembled genome, and obtain the genome assembly result of Aspergillus telomere to telomere.

9. A computer device, characterized in that, include: One or more processors; Memory, used to store one or more programs; When the one or more programs are executed by the one or more processors, the one or more processors implement the Aspergillus telomere-to-telomere genome assembly method as described in any one of claims 1-7.

10. A storage medium containing computer-executable instructions, characterized in that, The computer-executable instructions, when executed by a computer processor, are used to perform the Aspergillus telomere-to-telomere genome assembly method as described in any one of claims 1-7.

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