CN111445948A - Chromosome construction method for polyploid fish by using Hi-C - Google Patents
Chromosome construction method for polyploid fish by using Hi-C Download PDFInfo
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Abstract
A chromosome construction method for polyploid fishes by using Hi-C belongs to the technical field of molecular biology and comprises the following steps: 1) constructing contigs according to the contigs among the sequences; 2) splitting subgenomic based on whole genome alignment; 3) constructing a Hi-C library and sequencing the Hi-C library; 4) Hi-C constructs chromosomal sequences. According to the method, the chromosome sequence can be obtained only by preliminarily assembled polyploid genomes according to the combination of the sub-genome assembly and the Hi-C, so that an effective method is provided for the assembly of the chromosomes of polyploid fishes, and a feasible technology is provided for the functional genome research, genetic breeding and developmental evolution of the polyploid fishes.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a chromosome construction method for polyploid fish by using Hi-C.
Background
Two polyploid animals, including naturally evolved polyploids and artificially induced polyploids, have been widely used in global aquaculture, and many economically important aquaculture fishes, such as carps, salmon, carps and tunas, are natural polyploids or have evolved from polyploid progenitors. Polyploids and have three distinct advantages: heterosis, gene redundancy and unisexual reproduction, which may drive evolution and enrich species diversity, polyploidization may also lead to transient speciation.
Construction of chromosome-level assemblies is a key step in achieving the "platinum" genomic goal, but assembly and anchoring of sequences into polyploid chromosomes, especially fish polyploids, remains a significant challenge due to the high degree of similarity between polyploid-forming sequences. Previous techniques have used genetic maps, but genetic maps have poor discrimination and require the construction of isolated populations of up to about 100 individuals for sequencing. High throughput chromosome conformation capture (Hi-C) technology can significantly improve chromosome construction as a powerful tool, originally aimed at interrogating the 3D structure of the genome inside the nucleus by measuring the frequency of contacts between all pairs of loci in the genome. The frequency of contacts between a pair of loci is closely related to the one-dimensional distance between them, and the results indicate that Hi-C data can be used for chromosomal assembly of the genome.
The invention provides a new method, namely a chromosome construction method (FishHi-C) for polyploid fish by using Hi-C, which can construct contig by using the result of the sub-genome split by polyploid and using read comparison of Hi-C and the sequencing of comparison relation. The FishHi-C can be used for realizing the assembly of haploid chromosome level chromosomes of homologous polyploid fishes and heterozygous diploid genomes, and the obstacle of assembling complex genomes is overcome. Obtaining the high-quality chromosome-level polyploid fish assembly result, and providing solid support for genetic researches such as genetic breeding, developmental evolution research and the like of fish.
Disclosure of Invention
In order to realize the assembly of haploid chromosome level chromosomes of homologous polyploid fishes and heterozygous diploid genomes and overcome the obstacle of assembling complex genomes, the invention discloses a method for constructing the chromosomes of the polyploid fishes by using Hi-C, the method splits subgenomic groups according to the similarity of the polyploid subgenomic groups, and carries out chromosome linking according to Hi-C after the splitting is finished to obtain the polyploid chromosome level assembly result, and the specific technical scheme is as follows:
a chromosome construction method for polyploid fish by using Hi-C comprises the following steps:
selecting a long sequence to be 100-150 times of the depth of a genome, selecting a sequence with the read length being more than 8kb and the average mass value of the sequence being more than 10, and then constructing a preliminary assembly result, namely contig, by a WGD-based strategy; and selecting Flye assembly software for contig assembly, wherein the debugging parameters are as follows: -asm-coverage 60-pacbio-raw-t 90;
the constructed contigs were aligned genome-wide using L astz, the result was first masked using repeat annotation of repeat mask to prevent too many invalid alignments, and then aligned using L astz with alignment parameters tuned as-seed 12of 19-noise-chain-gapped-gap 400, 30-hsptres 2000-gapped-3000-ydrop 3400-gapped-4000-inner 2000-format axt-registers/motions/mapping/software/rock-trip-trib-1.04.00/bin/hoxd55. the longer of the two best aligned sequences was defined as subgenomic R1, the shorter of subgenomic R2, the alignment was not defined as subgenomic R1;
fixing the leaves in MS buffer solution with 1% formaldehyde solution at room temperature for 30 min; after fixation, putting the muscle tissue of the polyploid fish into MC buffer solution containing 0.15M glycine and incubating for 5-6 min in vacuum at room temperature; homogenizing muscle tissues of 2-3 g of polyploid fish by liquid nitrogen, suspending in MS buffer solution, filtering by a 40nm cell filter, and completing the process of enriching nuclei from circulation and subsequent denaturation according to a 3C procedure established for the fish;
digesting chromatin at 37 ℃ with 400UHind III restriction enzyme (NEB) for 16 hours, labeling DNA ends with biotin and incubating at 37 ℃ for 45min, then inactivating HindIII restriction enzyme with 20% SDS solution, performing DNA ligation by adding T4DNA ligase (NEB) and incubating at 16 ℃ for 4-6 hours, after ligation, adding proteinase K during incubation overnight at 65 ℃ to reverse crosslink, purifying and dissolving the DNA fragment in 86 μ L water, then removing unligated ends, purifying the DNA fragment with a size of 300-500bp, then repairing the DNA ends, finally, separating the biotin-labeled DNA fragment on Dynabeads M-280Streptavidin (L iftechnies), controlling the quality of the Hi-C library, and sequencing on an Illumina HiseqXTen instrument with a read length of 150 bp and insert fragment of 300 bp;
firstly, comparing 100X sequencing data of NGS to assembled contigs by using bowtie2, comparing parameters- -sensitive-fq1fq1. gz-fq2fq2. gz, selecting unique comparison from comparison results, checking the length distribution of insert fragments in pair comparison according to the comparison results, and selecting insert fragments with more than 1000 comparisons as effective comparison results; then calculating interaction frequency between different pairs, determining similar chemical fragments by using a clustering algorithm according to different interaction frequencies, drawing a heat map of frequency interaction, judging a plurality of large interaction areas according to the heat map, recording positions on the heat map, and clustering according to the positions to obtain a chromosome map; connecting the result of the chromosome map to the assembly result to obtain a chromosome sequence;
in the step 3, the MS buffer solution consists of 10mM potassium phosphate, pH 7.0; 50mM NaCl; 0.1M sucrose.
Compared with the prior art, the method for constructing the chromosome of the polyploid fish by using the Hi-C has the beneficial effects that:
firstly, the method creatively splits a polyploid subgenome, and then assembles the subgenome to a chromosome by utilizing the sequence interaction principle of Hi-C. All chromosome sequences of polyploid fish are obtained through the chromosome of the subgenome. Compared with 100 individuals of the genetic map, the method only needs one individual, and the sequencing data amount is reduced from 500 times to 100 times.
And secondly, the resolution is as high as 700 bases, and the chromosome integrity is more than 90% relative to 100 ten thousand bases of the genetic map, but the genetic map is usually only 60%.
Thirdly, the method obtains a preliminary assembly result for the polyploid sequencing of the fish at about 200 times of depth, then splits the subgenomic group, and then utilizes the subgenomic group and the Hi-C to solve the problem of complexity and high cost of the process of constructing the chromosome by using the genetic map of the polyploid fish, and the method has the resolution of up to 700 bases which is far more than 100 ten thousand bases of the genetic map.
Drawings
FIG. 1 is a diagram of a whole genome alignment split sub-genome according to an embodiment of the present invention;
FIG. 2 is a heat map of the frequency interactions on a subgenome of Hi-C sequence alignments according to an embodiment of the present invention;
FIG. 3 is a diagram of Hi-C sequencing, library building, sequencing and assembly according to an embodiment of the present invention: wherein, 1-chromosome and protein interaction region, 2-genome disruption, 3-biotin enrichment, 4-reconnection, 5-invalid data and 6-valid data.
Detailed description of the invention
The invention will be further described with reference to specific embodiments and figures 1-3, but the invention is not limited to these embodiments.
A chromosome construction method for polyploid fishes by using Hi-C specifically comprises the following steps:
selecting a long sequence to sequence at 100-fold genome depth, selecting a sequence with the read length larger than 8kb and the average mass value of the sequence larger than 10, and then constructing a preliminary assembly result, namely contig, by a WGD-based strategy; and selecting Flye assembly software for contig assembly, wherein the debugging parameters are as follows: -asm-coverage 60-pacbio-raw-t 90;
l astz for whole genome alignment, firstly using repeat annotation result of repeatmasker to mask genome, preventing excessive invalid alignment, then using L astz for alignment, the alignment parameters are adjusted as-seed 12of 19-mutation-chain-gain-gap 400, 30-hsptresh 2000-gappedtresh 3000-ydrop 3400-gappedtresh-4000-inner 2000-format axt-organs/home/fandingding/software/last-distribb-1.04.00/bin/hoxd55. scanner, here we select genome of anorthose schizothorax with genome size 2G, 50% of repeat region on genome, 48% of genome is removed, the comparison result is obtained after screened 48 hours, the comparison result is defined as sub-genome mapping result R96, the comparison result is defined as short R.3875, the comparison result is defined as R.6, the comparison result is not obtained according to the comparison factor R.3875, and the comparison result is defined as the comparison factor R.3875, which is not obtained according to the comparison result of direct mapping, and the comparison result is defined as the comparison factor R.38764;
the leaves were fixed with 1% formaldehyde solution in MS buffer (10mM potassium phosphate, pH 7.0; 50mM NaCl; 0.1M sucrose) for 30 minutes at room temperature; after fixation, the polyploid fish muscle tissue was incubated in MC buffer containing 0.15M glycine under vacuum for 5min at room temperature; 2g of- -asm-coverage60- -pacbio-raw-t90 were homogenized with liquid nitrogen, resuspended in MS buffer and filtered through a 40nm cell filter; the process of enriching nuclei from circulation and subsequent denaturation is completed according to the 3C protocol established for fish;
digesting chromatin at 37 ℃ with 400UHind III restriction enzyme (NEB) for 16 hours, labeling DNA ends with biotin and incubating at 37 ℃ for 45min, then inactivating HindIII restriction enzyme with 20% SDS solution, performing DNA ligation by adding T4DNA ligase (NEB) and incubating at 16 ℃ for 4-6 hours, after ligation, adding proteinase K during incubation overnight at 65 ℃ to reverse crosslink, purifying and dissolving the DNA fragment in 86 μ L water, then removing unligated ends, purifying the DNA fragment with a size of 300-500bp, then repairing the DNA ends, finally, separating the biotin-labeled DNA fragment on Dynabeads M-280Streptavidin (L iftechnies), controlling the quality of the Hi-C library, and sequencing on an Illumina HiseqXTen instrument with a read length of 150 bp and insert fragment of 300 bp;
firstly, comparing 100X sequencing data of NGS to assembled contigs by using bowtie2, comparing parameters- -sensitive-fq1fq1. gz-fq2fq2. gz, selecting unique comparison from comparison results, checking the length distribution of insert fragments in pair comparison according to the comparison results, and selecting insert fragments with more than 1000 comparisons as effective comparison results; then calculating the interaction frequency between different pairs, determining similar chemical fragments by using a clustering algorithm according to the difference of the interaction frequency, drawing a heat map of frequency interaction, judging a plurality of large interaction areas according to the heat map, recording the positions on the heat map, and then clustering according to the positions to obtain a chromosome map, wherein the heat map is shown in figure 2; connecting the result of the chromosome map to the assembly result to obtain a chromosome sequence; as shown in fig. 3.
Claims (3)
1. A chromosome construction method for polyploid fish by using Hi-C is characterized by comprising the following steps:
step 1, constructing contig according to the contig between sequences:
selecting a long sequence to be 100-150 times of the depth of a genome, selecting a sequence with the read length being more than 8kb and the average mass value of the sequence being more than 10, and then constructing a preliminary assembly result, namely contig, by a WGD-based strategy; and selecting Flye assembly software for contig assembly, wherein the debugging parameters are as follows: -asm-coverage 60-pacbio-raw-t 90;
step 2, splitting subgenomic based on whole genome comparison:
the constructed contigs were aligned genome-wide using L astz, the result was first masked using repeat annotation of repeat mask to prevent too many invalid alignments, and then aligned using L astz with alignment parameters tuned as-seed 12of 19-noise-chain-gapped-gap 400, 30-hsptres 2000-gapped-3000-ydrop 3400-gapped-4000-inner 2000-format axt-registers/motions/mapping/software/rock-trip-trib-1.04.00/bin/hoxd55. the longer of the two best aligned sequences was defined as subgenomic R1, the shorter of subgenomic R2, the alignment was not defined as subgenomic R1;
step 3, Hi-C library construction and sequencing:
fixing the leaves in MS buffer solution with 1% formaldehyde solution at room temperature for 30 min; after fixation, putting the muscle tissue of the polyploid fish into MC buffer solution containing 0.15M glycine and incubating for 5-6 min in vacuum at room temperature; homogenizing muscle tissues of 2-3 g of polyploid fish by liquid nitrogen, suspending in MS buffer solution, filtering, and completing the process of enriching nuclei from circulation and subsequent denaturation according to a 3C procedure established for the fish;
digesting chromatin at 37 ℃ with 400U HindIII restriction enzyme (NEB) for 16 hours, labeling DNA ends with biotin, incubating at 37 ℃ for 45min, then inactivating HindIII restriction enzyme with 20% SDS solution, performing DNA ligation by adding T4DNA ligase (NEB), incubating at 16 ℃ for 4-6 hours, adding proteinase K for reverse crosslinking after ligation, incubating overnight at 65 ℃, purifying and dissolving DNA fragments in 86 μ L water, then removing unligated ends, purifying DNA fragments with a size of 300 and 500bp, then repairing DNA ends, finally, separating the DNA fragments labeled with biotin on Dynabeads M-280 streptavidins (L Technologies), controlling the quality of Hi-C library, sequencing on Illumina Hiseq X Ten instrument, reading the length to 150 bp, and inserting the fragments to 300 bp;
step 4, Hi-C constructs a chromosome sequence:
firstly, comparing 100X sequencing data of NGS to assembled contigs by using bowtie2, selecting unique comparison according to comparison results, checking the length distribution of insert fragments on pair comparison according to the comparison results, and selecting insert fragments with more than 1000 comparison as effective comparison results, wherein the comparison parameters are sensory-fq 1fq1. gz-fq 2fq2. gz; then calculating interaction frequency between different pairs, determining similar chemical fragments by using a clustering algorithm according to different interaction frequencies, drawing a heat map of frequency interaction, judging a plurality of large interaction areas according to the heat map, recording positions on the heat map, and clustering according to the positions to obtain a chromosome map; and connecting the result of the chromosome map to the assembly result to obtain the chromosome sequence.
2. The method for chromosome construction of polyploid fish according to claim 1, wherein the MS buffer solution has the composition of 10mM potassium phosphate, pH 7.0; 50mM NaCl; 0.1M sucrose.
3. The method for chromosome construction of polyploid fish with Hi-C according to claim 1, wherein in step 3, the filtration uses a 40nm cell filter.
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