misMM: An Integrated Pipeline for Misassembly Detection Using Genotyping-by-Sequencing and Its Validation with BAC End Library Sequences and Gene Synteny

Article information

Genomics Inform. 2017;15(4):128-135
Publication date (electronic) : 2017 December 29
doi : https://doi.org/10.5808/GI.2017.15.4.128
1Department of Bioinformatics and Life Science, Soongsil University, Seoul 06978, Korea
2Genomics Division, Department of Agricultural Biotechnology, National Institute of Agricultural Sciences, Rural Development Administration, Jeonju 54874, Korea
*Corresponding author: Tel: +82-2-820-0457, Fax: +82-2-824-4383, E-mail: sskimb@ssu.ac.kr
Received 2017 October 24; Accepted 2017 November 2.


As next-generation sequencing technologies have advanced, enormous amounts of whole-genome sequence information in various species have been released. However, it is still difficult to assemble the whole genome precisely, due to inherent limitations of short-read sequencing technologies. In particular, the complexities of plants are incomparable to those of microorganisms or animals because of whole-genome duplications, repeat insertions, and Numt insertions, etc. In this study, we describe a new method for detecting misassembly sequence regions of Brassica rapa with genotyping-by-sequencing, followed by MadMapper clustering. The misassembly candidate regions were cross-checked with BAC clone paired-ends library sequences that have been mapped to the reference genome. The results were further verified with gene synteny relations between Brassica rapa and Arabidopsis thaliana. We conclude that this method will help detect misassembly regions and be applicable to incompletely assembled reference genomes from a variety of species.


The genomics era has opened in earnest with the completion of the Human Genome Project. With the development of next-generation sequencing (NGS) technologies, the amount of genomics data has exploded, and sequencing targets have become very diverse. As of 2017, there are 7,930 species of eukaryotes, 192,677 species of bacteria, and 1,412 species of archaea that have been officially registered in NCBI. As the Nagoya Protocol is initiated, it is expected that these numbers will continue to increase in the future due to the policies of each country to secure information on biological genetic resources [1, 2]. Despite the fact that the cost of genomic analysis is declining, there are still a number of technical problems that make it difficult to sequence the genome completely [3]. For example, misassembly due to the inherent limitations of NGS technology is well known [46]. Especially in plants, there are many barriers that make plant genomes hard to sequencing, such as Numts, repeats, and genome duplication events [79].

Genotyping-by-sequencing (GBS) is a technology that allows high-throughput genotyping by applying NGS technology. It is used to analyze single nucleotide polymorphisms (SNPs) in populations to find molecular markers that are related to phenotype and genotype or to draw genetic linkage maps for plant breeding. By analyzing the pattern of GBS data along each chromosome, one can find out where the gene crossover occurs. On the other hand, a small block that interrupts an otherwise continuous GBS pattern is genetically non-ideal and implies a misassembled region. Therefore, we explored the application of GBS in the detection of misassemblies [1012].

Brassicaceae is a mustard family containing 372 genera and 4,060 accepted species, and its varieties are cultivated as economically valuable crops not only in East Asia but also globally [13]. The triangle of U theory states that the differentiation of an allotetraploid of Brassica species—Brassica juncea (AABB), Brassica napus (AACC), and Brassica carinata (BBCC)—occurs due to the polyploidization of diploid Brassica species: Brassica rapa (AA), Brassica nigra (BB), and Brassica oleracea (CC). This theory has been proven by genomic analysis by NGS of Brassica species [1425]. Research on the correlation between the genetic information and the nutrient content of crops has been actively conducted in Brassica genomes [26]. The recently published B. rapa V2.1 genome sequence shows much improved quality, as well as a number of misassembly corrections over the previous version, V1.5 [17]. This offers an interesting opportunity to test the potential of misassembly detection, based on GBS data.

In this study, we propose a user-friendly pipeline, called misMM, which automatically identifies misassembled candidate blocks (MCBs) and adjacent to destination blocks (ADBs) and plots the genetic map of MCBs by using raw GBS data sorted by MadMapper [27]. These results are verified by using the BAC end-sequence library published in NCBI and the gene synteny relation between Arabidopsis thaliana and B. rapa [2831].


Data source

The end sequences of B. rapa accession Chiifu-401–42, a Chinese cabbage BAC library (KBrH, KBrB, and KBrS), were downloaded from NCBI and used to verify the putative misassembly genome regions. In order to investigate the gene synteny relation between A. thaliana TAIR10 and B. rapa genome V1.5, the corresponding general feature format (GFF) annotation files and protein sequences of each species were downloaded from http://ensemblgenomes.org and http://brassicadb.org, respectively. The GBS data were produced by a previous study that investigated the correlation between flavonoid content and the genotype of B. rapa in 69 individuals of a doubled haploid F2 generation obtained by microbial culture of an F1 generation cross of two subspecies—yellow sarson of LP08 (B. rapa ssp. tricolaris) and pak choi of LP21 (B. rapa ssp. chinensis)—with distinct morphologies [26]. From the study, genotype data were obtained at a total of 8,176 positions.

Configuration of the misMM pipeline for misassembled block detection

misMM, a pipeline for genome misassembled block detection, was written in a Linux shell and with Python ver. 2.7 in-house codes. The first step is preprocessing: after loading all GBS raw data files, markers with a missing value of over 8% were filtered out. If the neighboring positions had the similar GBS pattern with consistency, they were grouped into one block. Our script then automatically prepared the three kinds of input files (.loc, IDs, and maps) for MadMapper (UC Davis) [27], a package that specializes in recombinant inbred lines analysis using large genetic markers and easy visualizes the 2D pairwise matrix. The next step is the linkage grouping and block shuffling step, performed with MadMapper. By using the default parameters of MadMapper_RECBIT (rec_cut, 0.2; bit_cut, 100; data_cut, 25; allele_dist, 0.33; missing_data, 50; trio_analysis, TRIO; double_cross, 3), linkage grouping and marker extraction were performed by generating a pairwise matrix between GBS patterns of each block. Subsequently, block shuffling was performed by MadMapper_XDELTA (marker fixation, FIXED; shuffle option, SHUFFLE; shuffle block, 6; shuffle step, 3) with each clustered block. At the end of this process, it plotted a genetic map diagram with putative misassembled blocks. In addition, it also generated 2D heatmap graphs for comparing before and after the block shuffling. All of the work flow of this pipeline is described in Fig. 1. The misMM pipeline scripts can be downloaded from http://sskimbnas.ipdisk.co.kr:80/publist/HDD1/misMM/misMM.tar.gz.

Fig. 1

Total work flow of misMM. GBS, genotyping-by-sequencing.

Validation using BAC end sequences

In order to confirm the misassembled blocks with experimental data, we extracted 41,969 pairs of end sequences from the BAC libraries (KBrS, KBrH, and KBrB) of B. rapa and carried out sequence alignment against the B. rapa reference genome sequence using Nucmer (MUMmer3.23) with the proper options (--maxmatch, use all anchor matches; -g, global alignment; -I, >95%; -r, sort output lines by reference). The Nucmer results were then filtered for discordant BAC end pairs with one end aligned to the MCB and the other end to the ADB.

Validation using gene synteny relation between A. thaliana and B. rapa

For validation with gene synteny, the protein sequence of B. rapa were matched to those of A. thaliana using BLASTP (Blast 2.2.26), and the top four hits for each query were retained. The tabulated results were then sorted, based on the genomic coordinates of each protein, and the gene synteny relation was examined manually.

Results and Discussion

misMM was developed to provide a streamlined and yet simple-to-use pipeline for the detection of misassembled regions, so-called MCBs, based on GBS data (Fig. 1). This pipeline was tested with the GBS data of B. rapa against the B. rapa V1.5 reference genome, which is known to have some misassembled regions compared to the recently published V2.1 genome [17]. The original linkage score heatmap that was produced by MadMapper showed many off-diagonal cells with a low score that were often clustered in stretch (Fig. 2 left panel). The off-diagonal blocks scoring less than 0.33 were defined as MCBs (Table 1, Fig. 3). For each MCB, the corresponding ADB was identified by MadMapper, based on the linkage score (Table 1). The subsequent shuffled heatmap showed clean clustering, with no low-scoring off-diagonal blocks, implying the unambiguousness of the GBS pattern in detecting misassemblies (Fig. 2 right panel). The MCBs and ADBs were distributed throughout the entire pseudomolecule. A total of 16 MCBs had an average block size of 65,477 bp, and the largest one was 410,190 bp. The average size of the ADBs was 746,707 bp, with a maximum of 4,936,893 bp. The fact that only a few small MCBs were detected and that the corresponding ADBs were large in size implies that the B. rapa V1.5 genome is well assembled overall but has a few problematic regions, as shown by the recent update of the genome [17].

Fig. 2

Before and after the results of the 2D matrix graphs of the MadMapper block shuffling analysis. A01 through A10 indicate the Brassica rapa pseudomolecules.

Results of misassembled block detection analysis in Brassica rapa with misMM

Fig. 3

Example of Brassica rapa genetic map made with misMM pipeline. Red colors indicate misassembled candidate blocks.

We used two sets of data to validate that the ADBs were indeed in the neighboring area of the MCBs. The first one was used to find discordant BAC end pairs with one end aligned to the MCB and the other end aligned to the ADB. For example, the MCB of block number 2 in Table 1 was located in pseudomolecule A01, ranging from 11,453,104 to 11,488,588, while its corresponding ADBs were found in A04. Table 2 shows the mapping results of the six BAC end pairs of this block, the sizes of which ranged from 671 bp to 1,000 bp, with a mapping identity higher than 97.93%. While one end of the BAC pairs was mapped to the corresponding MCB in A01, all of the other ends were mapped within the ADB, ranging from 3,271,457 to 4,978,203 in A04. Likewise, 10 out of 16 blocks listed in Table 1 could be confirmed by the BAC end results. The true locations of these blocks could be estimated within the span of the corresponding BAC (average 110 kbp). The rest could not be confirmed, probably due to the distance between the MCB and ADB, making it incompatible with the BAC size.

Example of validation of BAC end library results

The other validation method was the use of the gene synteny relation. Compared to the A. thaliana genome, there is evidence that the B. rapa genome has undergone triplication [32]. Accordingly, most of the A. thaliana genes are preserved in gene synteny blocks at three different places. Within block number 2 in Table 1, two B. rapa genes are annotated: Bra033489 and Bra033490 (Table 3). For all 16 genes flanking these two genes, orthologs were identified by BLASTP (Table 4). Eight A. thaliana genes in the middle—including the orthologs of two genes, AT4G14330 and AT4G14350—were out of order and broke the continuity of the synteny in the region. This is consistent with our finding that this MCB is truly misplaced in B. rapa genome V1.5. The true locations of the two B. rapa genes in this MCB can be inferred by mapping the flanking genes of AT4G14330 and AT4G14350 to the B. rapa genome (Table 5). Indeed, a total of six A. thaliana flanking genes were mapped to the B. rapa orthologs that were found in the corresponding ADBs. As expected, the gene synteny of this region is also well preserved. In this way, we can estimate the approximate relative locations of these two genes. Based on this relationship, an analysis was carried out with regard to the relationship of the protein orthologs and gene coordination between the two species. First, two genes were annotated in an example block (Table 3). When these two genes were found in a table arranged by the coordinates of the B. rapa gene, there was no continuity between the ortholog genes and the surrounding genes (Table 4). But, when we sorted this based on the coordination of A. thaliana, the ortholog genes belonging to the ADB were located consecutively around the gene belonging to the MCB (Table 5). Furthermore, the gene order that was inferred here was confirmed in the updated B. rapa V2.1 genome that was recently published [17].

Information on genes included in example misassembled candidate block

Example of protein ortholog list, sorted by Brassica rapa gene coordination

Example of protein ortholog list, sorted by Arabidopsis thaliana gene coordination

In recent years, studies of expression quantitative trait loci that affect mRNA expression or protein expression using SNPs and studies to find markers that affect the environmental adaptation of plants have been becoming widely embraced [33]. For such works, accurate reference genome assembly is required. Toward that goal, our misMM pipeline is a useful tool for the identification of misassemblies in complex genomes using GBS data.


This work was funded by a program (PJ01167402) of the RDA (Rural Development Administration) and a program (NRF-2012M3A9D1054705) of the NRF (National Research Foundation of Korea) and computationally supported by a program (NRF-2010-0018156, NTIS1711048528) of the KISTI GSDC.


Authors’ contribution

Conceptualization: YJK, JSK

Data curation: YJK

Formal analysis: YJK

Funding acquisition: SK

Methodology: YJK

Writing – original draft: YJK

Writing – review & editing: SK, JSK


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Article information Continued

Fig. 1

Total work flow of misMM. GBS, genotyping-by-sequencing.

Fig. 2

Before and after the results of the 2D matrix graphs of the MadMapper block shuffling analysis. A01 through A10 indicate the Brassica rapa pseudomolecules.

Fig. 3

Example of Brassica rapa genetic map made with misMM pipeline. Red colors indicate misassembled candidate blocks.

Table 1

Results of misassembled block detection analysis in Brassica rapa with misMM

Block No. Misassembled candidate block Adjacent to destination block Synteny relation Count of BAC end

Chr No. Start position End position Block size (bp) Chr No. Start position End position Block size (bp)
1 A01 10,335,503 10,336,457 955 A07 2,718,763 2,760,427 41,665 No gene 1
2,970,361 3,340,395 370,035
4,284,326 5,685,009 1,400,684
5,782,516 8,114,350 2,331,835
8,306,623 8,390,460 83,838
8,462,236 9,063,378 601,143

2 A01 11,453,104 11,488,558 35,455 A04 3,271,457 4,978,203 1,706,747 Related 6
5,227,803 6,734,498 1,506,696
7,605,871 7,605,928 58

3 A01 11,830,981 - 1 A05 10,274,396 14,490,617 4,216,222 Related 1
A07 13,576,261 - 1 14,602,065 14,704,957 102,893
A08 1,389,252 1,419,543 30,292 14,946,890 15,735,698 788,809
6,968,479 7,090,412 121,934
7,231,217 7,782,948 551,732
7,825,594 8,040,473 214,880
8,683,679 9,511,317 827,639

4 A01 17,853,386 17,853,417 32 A03 28,233,583 28,599,515 365,933 Related 2
A01 21,422,470 21,756,693 334,224 28,622,787 29,191,693 568,907
A02 26,385,973 26,386,023 51 29,806,067 31,527,446 1,721,380

5 A01 23,266,604 23,424,555 157,952 A06 10,280,840 10,357,155 76,316 Related 13
A02 13,440,136 13,440,137 2 10,732,633 14,236,176 3,503,544
A02 21,066,162 21,066,274 113 14,450,457 14,559,524 109,068
8,950,753 10,162,388 1,211,636

6 A01 8,706,169 8,950,670 244,502 A09 11,293,419 11,528,445 235,027 Related 63
A06 19,457,789 19,703,630 245,842 11,610,344 14,668,929 3,058,586
14,915,372 15,794,376 879,005
15,949,568 18,361,629 2,412,062
19,064,188 22,487,944 3,423,757
22,634,782 23,337,316 702,535
23,489,828 23,489,836 9

7 A02 21,427,161 - 1 A10 11,579,416 - 1 No gene 0
176,000 1,638,829 1,462,830
1,765,780 1,766,618 839
1,786,668 1,792,156 5,489
3,686,351 5,224,789 1,538,439
5,335,436 5,459,255 123,820
5,648,752 5,693,352 44,601

8 A03 15,343,238 - 1 A08 2,368,697 3,803,367 1,434,671 Related 2
4,037,929 4,357,798 319,870
4,787,505 4,835,708 48,204
5,188,553 6,046,948 858,396
7,117,019 7,501,164 384,146
7,559,836 8,722,062 1,162,227
9,000,508 9,002,057 1,550

9 A05 8,144,773 8,162,600 17,828 A08 19,141,593 19,320,715 179,123 Related 16
8,217,883 8,234,265 16,383 19,394,784 19,497,679 102,896
8,250,451 8,352,384 101,934 19,568,112 19,621,358 53,247
19,674,213 19,711,491 37,279

10 A05 9,669,449 10,079,638 410,190 A01 10,376,926 10,494,252 117,327 Related 23
10,687,155 11,394,090 706,936
11,519,211 11,744,579 225,369
11,900,084 16,836,976 4,936,893
16,848,291 17,125,316 277,026
17,226,336 17,789,202 562,867
17,860,877 18,575,071 714,195
9,305,241 9,707,259 402,019
9,711,107 10,267,937 556,831

11 A07 2,319,220 2,321,114 1,895 A01 24,324,484 24,353,432 28,949 Related 0
24,402,955 24,488,344 85,390
24,619,832 24,806,034 186,203
24,920,288 24,920,419 132

12 A07 3,920,950 4,009,069 88,120 A10 11,579,416 1 Related 0
A08 3,927,665 - 1 176,000 1,638,829 1,462,830
1,765,780 1,766,618 839
1,786,668 1,792,156 5,489
3,686,351 5,224,789 1,538,439
5,335,436 5,459,255 123,820
5,648,752 5,693,352 44,601

13 A07 8,271,542 8,274,604 3,063 A03 12,032,914 12,032,953 40 Related 5
12,049,203 12,406,487 357,285
12,473,776 13,917,498 1,443,723
14,019,224 14,200,642 181,419
14,222,379 14,355,939 133,561

14 A08 11,266,789 - 1 A03 25,996,840 26,033,638 36,799 Related 0
26,067,147 27,037,677 970,531
27,139,966 27,943,662 803,697

15 A05 8,552,907 8,593,005 40,099 A02 11,596,619 13,185,910 1,589,292 Related 0
A08 1,584,456 1,594,851 10,396 13,498,575 14,449,253 950,679
14,804,284 18,303,089 3,498,806
18,558,399 19,378,431 820,033
19,548,342 19,666,145 117,804
19,787,176 1

16 A08 4,941,300 4,969,852 28,553 A10 11,146,660 11,437,447 290,788 Related 0
11,664,229 1
11,764,627 11,814,368 49,742
11,928,253 12,032,475 104,223

Table 2

Example of validation of BAC end library results

BAC ends library ID gi No. Length (bp) Identity (%) Brassica rapa
Chr No. Start position End position
KBrB037L22F 84732862 671 97.93 A01 11,474,904 11,475,144
KBrB037L22R 84732863 671 99.4 A04 4,869,416 4,870,085
KBrB039C19R 84733951 869 99.65 A01 11,471,320 11,472,188
KBrB039C19F 84733950 822 99.76 A04 4,884,036 4,884,855
KBrB043O24F 84737591 874 99.89 A01 11,452,951 11,453,822
KBrB043O24R 84737592 816 100 A04 4,884,025 4,884,840
KBrB077H15F 84762968 617 98.92 A01 11,474,904 11,475,088
KBrB077H15R 84762969 646 100 A04 4,884,386 4,885,031
KBrB097P17F 114827207 1,000 98.2 A01 11,471,303 11,472,294
KBrB097P17R 114827208 937 98.16 A04 4,883,252 4,884,169
KBrH087A11R 84341421 831 99.88 A01 11,466,761 11,467,587
KBrH087A11F 84341072 844 99.63 A04 4,977,838 4,978,643

Table 3

Information on genes included in example misassembled candidate block

Chr No. Type Start point End point Brassica rapa ID
A01 Gene 11,455,026 11,470,735 Bra033489
A01 Gene 11,451,545 11,454,600 Bra033490

Table 4

Example of protein ortholog list, sorted by Brassica rapa gene coordination

Brassica rapa Arabidopsis thaliana Comments

ID Chr No. Start position End position ID Chr No. Start position End position
Bra033497 A01 11,382,249 11,386,827 AT4G15570 Chr4 8,892,607 8,898,999 -

Bra033496 A01 11,388,925 11,390,027 AT4G15563 Chr4 8,890,879 8,892,526 -

Bra033495 A01 11,393,659 11,396,663 AT4G15560 Chr4 8,883,907 8,887,565 -

Bra033494 A01 11,410,610 11,412,043 AT4G15550 Chr4 8,877,590 8,879,327 -

Bra033493 A01 11,412,702 11,414,443 AT4G15545 Chr4 8,875,918 8,877,799 -

Bra033492 A01 11,445,862 11,446,743 AT5G49420 Chr5 20,034,674 20,036,170 -

Bra033491 A01 11,450,091 11,451,172 AT4G14320 Chr4 8,241,732 8,243,910 -

Bra033490 A01 11,451,545 11,454,600 AT4G14330 Chr4 8,244,194 8,247,444 Misassembled candidate

Bra033489 A01 11,455,026 11,470,735 AT4G14350 Chr4 8,256,086 8,260,787 Misassembled candidate

Bra039534 A01 11,504,946 11,505,630 AT2G35280 Chr2 14,859,378 14,860,200 -

Bra039535 A01 11,504,946 11,505,422 AT2G35280 Chr2 14,859,378 14,860,200 -

Bra039536 A01 11,504,994 11,505,630 AT2G35280 Chr2 14,859,378 14,860,200 -

Bra039538 A01 11,510,855 11,512,648 AT3G59380 Chr3 21,944,178 21,945,943 -

Bra039539 A01 11,514,776 11,515,144 AT4G15530 Chr4 8,864,828 8,870,967 -

Bra039540 A01 11,516,583 11,521,200 AT4G15530 Chr4 8,864,828 8,870,967 -

Bra039541 A01 11,521,728 11,523,067 AT4G15520 Chr4 8,862,815 8,864,618 -

Table 5

Example of protein ortholog list, sorted by Arabidopsis thaliana gene coordination

Brassica rapa Arabidopsis thaliana Comments

ID Chr No. Start position End position ID Chr No. Start position End position
Bra032781 A04 4,968,584 4,971,945 AT4G14290 Chr4 8,225,481 8,230,281 Included in ADB

Bra032782 A04 4,962,259 4,963,651 AT4G14305 Chr4 8,235,093 8,236,715 Included in ADB

Bra033490 A01 11,451,545 11,454,600 AT4G14330 Chr4 8,244,194 8,247,444 Included in MCB

Bra033489 A01 11,455,026 11,470,735 AT4G14350 Chr4 8,256,086 8,260,787 Included in MCB

Bra033487 A04 4,917,814 4,918,407 AT4G14380 Chr4 8,285,766 8,286,772 Included in ADB

Bra033486 A04 4,915,949 4,917,119 AT4G14385 Chr4 8,286,986 8,288,800 Included in ADB

Bra033483 A04 4,882,642 4,883,358 AT4G14440 Chr4 8,306,745 8,307,753 Included in ADB

Bra033482 A04 4,873,477 4,873,797 AT4G14450 Chr4 8,309,474 8,310,058 Included in ADB

Alternative alignments due to genome triplication have been removed.

ADB, adjacent to destination block; MCB, misassembled candidate blocks.