African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12487

Full Length Research Paper

Whole-genome optical mapping: Improving assembly of Macrophomina phaseolina MS6 through spanning of twelve blunt end chromosomes by obviating all errors and misassembles

Quazi Md. Mosaddeque Hossen
  • Quazi Md. Mosaddeque Hossen
  • Basic and Applied Research on Jute (BARJ) Project, Bangladesh Jute Research Institute, Dhaka, Bangladesh.
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Md. Shahidul Islam
  • Md. Shahidul Islam
  • Basic and Applied Research on Jute (BARJ) Project, Bangladesh Jute Research Institute, Dhaka, Bangladesh.
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Emdadul Mannan Emdad
  • Emdadul Mannan Emdad
  • Basic and Applied Research on Jute (BARJ) Project, Bangladesh Jute Research Institute, Dhaka, Bangladesh.
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Md. Samiul Haque
  • Md. Samiul Haque
  • Basic and Applied Research on Jute (BARJ) Project, Bangladesh Jute Research Institute, Dhaka, Bangladesh.
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Md. Monjurul Alam
  • Md. Monjurul Alam
  • Basic and Applied Research on Jute (BARJ) Project, Bangladesh Jute Research Institute, Dhaka, Bangladesh.
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Maqsudul Alam
  • Maqsudul Alam
  • Basic and Applied Research on Jute (BARJ) Project, Bangladesh Jute Research Institute, Dhaka, Bangladesh.
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  •  Received: 07 October 2019
  •  Accepted: 08 November 2019
  •  Published: 30 November 2019

 ABSTRACT

Deciphering genetic information through next-generation sequencing (NGS) is considered as the basic platform to unveil in details of an organism. However, as it produces short reads that lead to difficulties in assembly, we generated long scaffold-based optical mapping (OM) data of previously sequenced devastating fungus, Macrophomina phaseolina MS6. In the process, KpnI identified as the most effective restriction endonuclease among tested 13, used to digest high molecular weight (HMW) DNA that generated 270,343 genomic DNA molecules size in more than 250 kb. The molecules were assembled and constructed 12 super-scaffolds (terminated with telomeric blunt-ends and denoted as chromosomes) that were aligned with NGS generated 17 (out of 88 reduced from 94) reference scaffolds. The state-of-the-art technology revealed concordances and different discordances viz., inversions, low-quality assembly, gaps, overlaps followed to correct the NGS misassembles. Based on the results, OM generated improved and validated assembly advance our understanding of the chromosome evolution of fungi. This furnished data might be considered as valuable resources to accelerate the precise planning for the protection of M. phaseolina MS6 infected sequenced crops through developing the cross-talk phenomenon between the host and pathogen.
 
Key words: Macrophomina phaseolina MS-6, optical mapping, assembly improvement, assembly validation.


 INTRODUCTION

Genome sequencing is the process of determining the full DNA sequences of living organisms including its chromosomal, mitochondrial and chloroplast DNA. Although DNA sequencing seemed not to be an easier and faster process, the rapid development of different pipelines and techniques made the whole genome sequences simple over the last few years (Koboldt et al., 2013). However, NGS produces a large number of short reads restraints the de novo assembly due to repeat or complex region of genome that suffers extensive misassembles and comprise gaps (Pendleton et al., 2015;  Ganapathy  et  al.,  2014;   Ruperao   et al., 2014). Therefore, the demand of introducing a new technique was a must to minimize these errors in terms of whole-genome sequencing.
 
Whole-genome optical mapping, the cutting edge technology offered for resolving the issues through estimating the gap length between the scaffolds and merges them into much longer sequences without introducing new bases (Ghurye  and Pop, 2019; Kremer et al., 2017; Zhou et al., 2009; Aston et al., 1999; Samad et al., 1995). It also provides a valuable template for de novo genomic sequence assembly where large structural variations in the genome can accurately be detected and quantified (Long et al., 2018; Mak et al., 2016; Shukla et al., 2009; Teague et al., 2010). Furthermore, OM is capable of producing high-resolution, ordered, high-throughput genomic map data that gives information about the structure of a genome (Mukherjee et al., 2018; Schwartz et al., 1993). Though initially, it has been used to construct whole-genome restriction maps of bacteria, parasites, and fungi (Lai et al., 1999; Lim et al., 2001; Lin et al., 1999), recently it has been used for scaffolding contigs and for assembly validation of large-scale sequencing projects including maize, goat and Amborella genomes (Chamala et al., 2013; Udall and Dawe, 2018; Dong et al., 2013).
 
In Bangladesh, Jute (Corchorus species) is the most important cash crop considered the second foreign earning resources of the country (BBS, 2011). However, this crop is affected by several pathogens and diseases throughout its growing season and causing severe yield losses (Mamun et al., 2016). Among different agents Macrophomina phaseolina MS6, an ascomycetous, necrotrophic, soil-borne fungi, can solely reduce its yield up to 30% (Islam et al., 2012). This pathogen has more than 500 hosts (Lodha and Mawar, 2019; Khan et al., 2017; Islam et al., 2012) including major crops like cotton (Aly et al., 2007), jute (Meena et al., 2015; De et al., 1992), groundnut (Islam et al., 2012), maize (Biemond et al., 2013), sorghum (Su et al., 2001), millet (Lodha and Mawar, 2019), potato (Abbas et al., 2013), sesame (Dinakaran and Mohammed, 2001), soybean (Wyllie, 1993), beans (Mayek-Pérez et al., 2001), sunflower (Khan, 2007), sweet potato (Da Silva and Clark, 2013), tomato (Hyder et al., 2018), and tobacco (Wyllie, 1998). It outbreaks as stem rot (Majumder et al., 2018), seedling blight (Lu et al., 2015), charcoal rot (Majumder et al., 2018), dry root rot (Živanov et al., 2019), wilt (Piperkova et al., 2016), leaf blight (Mahadevakumar and Janardhana, 2016), pre and post-emergence damping-off (Hai et al., 2017), root and stem rot of softwood forest and fruit trees and also in weed species (Singh et al., 1990; McCain and Scharpf, 1989). This fungus forms microsclerotia in the soil and survives up to 15 years without attacking hosts (Kaur et al., 2012). It can also live in extreme environmental conditions like high temperature (30-35°C), low soil moisture, diverse pH, wide-ranging salt state and drought situation (Mengistu et al., 2011).
 
Considering all aforementioned consequences especially in jute, its genome was sequenced previously to have its mechanisms of attacking crops (Accession: AHHD00000000). But as described earlier, the bottlenecks that suffered the whole genome sequencing project, we addressed optical mapping to furnish and improve the genome. The furnished assembly was considered as valuable resources that might be used to develop a logical strategy for controlling the pathogen by unveiling the host-pathogen interaction within all sequenced crops that are infected by the pathogen.


 MATERIALS AND METHODS

Preparation of working sample from M. phaseolina MS6
 
The strain M. phaseolina MS6 was taken from a stem rot infected jute plant (Corchorus capsularis L). The pathogen was cultured and purified on Potato Dextrose (PD) media maintaining 30°C for 72 h in dark conditions. The grey-brown mycelia were collected and washed with physiological buffer (Na2HPO4, pH 7.0: NaH2PO4.H2O, pH <7.0) followed by drying under laminar flow Hood (Islam et al., 2012). 
 
Extraction of megabase size DNA
 
Spheroplasting
 
Two grams of mycelia was ground into fine powder in liquid nitrogen with a mortar and pestle and immediately transferred into an ice-cold 1000 ml beaker containing 800 to 1000 ml ice-cold 1x Homogenize Buffer (HB) (0.1 M Tris, 0.8 M KCl, O.1 M EDTA, 10 mM Spermidine, 10 mM Spermine) with 0.15% beta-mercaptoethanol and 0.5% Triton X-l00. The contents were swirled gently for 10 minutes on ice and filtered by two layers of cheesecloth followed to one layer of Miracloth (Sigma-Aldrich, USA). The homogenate was taken into a centrifuge to have the pellet with a fixed-angle rotor at 1,800 g at 4°C for 20 minutes. The supernatant was discarded and approximately 1 ml of ice-cold Wash Buffer (WB) (1x HB, 20% TritonX-100, 0.15% beta-mercaptoethanol) was added to each tube. The pellet was resuspended gently with a small paintbrush soaked in ice-cold wash buffer. The nuclei were pelleted by centrifugation at 1,800 g at 4°C for 15 minutes in a swinging bucket centrifuge. The pellets were washed additional three times by resuspending in washing buffer using a paintbrush followed by centrifugation at 1,800 g at 4°C for 15 minutes. The pelleted nuclei were resuspended again in a small amount (1 ml) of 1x HB without beta-mercaptoethanol followed by counting the nuclei (approx. 5 × 107 nuclei/ml), under the contrast phase microscope with addition of the 1x HB without beta-mercaptoethanol and stored on ice.
 
Embedding cells
 
Low-melting-point (LMP) agarose (1%) was prepared in 1x HB without beta-mercaptoethanol and Triton X-100 followed by cooling down to 45°C and maintained in a 45°C water bath before use. The nuclei were pre-warmed to 45°C in a water bath (5 minutes) and mixed with an equal volume of the pre-warmed 1% LMP agarose in 1x HB without beta-mercaptoethanol and Triton X-100 using a cut-off pipette tip. The mixture was aliquoted into ice-cold plug molds on ice with the same pipette tip at 100 ml per plug.
 
Lysis
 
The solidified gel was sliced into pieces and incubated in 50 ml of digestion buffer (0.5 M EDTA, 7.5% β-mercaptoethanol) at 37°C for overnight. The buffer was replaced with NDSK buffer (0.5 M EDTA, 1% (w/v) N-lauroylsarcosine, 1 mg/ml proteinase K) for downstream work.
 
Plugs washing
 
The plugs were placed in a new 50 ml conical tube and added 45 ml of 1X TE buffer. The conical tube was capped with a clean green sieve and a regular cap and rock on a platform rocker at low speed for 1 h. 1X TE buffer was decanted from the conical tube and added fresh 1X TE buffer followed by rocking on a platform rocker at low speed for another 1 h. It was repeated for three times.
 
Melting plugs
 
The plugs were transferred into a sterile Petri-dish and cut using a sterile scalpel in half and transferred to a separate 2.0 ml microcentrifuge tube. The microcentrifuge tubes were taken into a heat block at previously maintained 70°C for 7 minutes followed by pipetting 50 μl of the pre-warmed β-Agarose-TE solution (mixing 48 μl of 1X TE with 2 μl of 1 U/μl β-Agarose). The tube was incubated at 42°C heat block for overnight. Loading buffer was added to the DNA solution and stored at 4°C.
 
Restriction enzyme selection
 
The Enzyme.pl script (In-house script) was used to select optimal restriction endonuclease that generates restriction fragment statistics for different restriction enzymes. The optimal restriction enzyme was selected using this script by considering average fragment size (kb), fragment greater than 100 kb, maximum fragment size and the highest percentage of average fragment size underlie within 5 to 20 kb size fragments. 
 
MapCard setup and data collection
 
The high molecular weight DNA was placed on optical chips to make them linear. This immobilized DNA was digested randomly with a restriction endonuclease and subsequently stained with jojo-1TM dye (Life Technologies Corporation, USA) for image capturing and fragment size measurement. The mapset (total data sets generated from all runs) was put together for assembly by using the Argus system embedded Gentig map assembler to create a consensus optical map. The mapsets were considered for assembly after filtering them following minimum molecule length (>150 kb) with minimum fragments per molecule (>12) and minimum molecule quality score (0.2).
 
Optical mapping assembly
 
The data from each MapCard were combined for the final assembly. In the assembly process, the filtered mapsets were taken for aligning them to form contigs by overlapping and keeping some extending fragments for resolving both side blunt ends (telomeric end protection). The final restriction maps were obtained by fulfilling criteria like coverage, depth, genome complexity of primary and final draft contigs. Different errors like low occurrence, low depth, and potential misassembles, as well as potential problems like false cut, missing cut, and false fragments were obviated  using  find  hits techniques or removing the errors through Argus optical mapping embedded Gentig software packages. Assembly was conducted also with the removal of default circularization parameters. Partial assembly results were saved when 12 contigs became apparent by having >50 molecules each. Contigs were split off and reassembled against the original mapset individually using the "Find Hits" feature. Contigs were considered "finished" when no additional molecules were added by subsequent reassembles. Chromosome ends that were not blunt were visually inspected and any questionable molecule was removed from the final map (Figure 1).
 
 
 
Alignment by MapSolver
 
MapSolver software uses a dynamic programming algorithm to find the optimal placement location of each supplied sequence scaffolds in the Optical Map. The algorithm applies user-provided settings toward generating local alignments between each scaffold and the optical map. Scaffolds are aligned in both forward and reverse directions. MapSolver determines an alignment score for each comparison, where a higher score implies greater confidence in the alignment. Alignments with scores that meet or exceed the minimum score for local parameters are evaluated for placement. The number of aligned fragments must also meet or exceed the number specified by the Minimum aligned fragments parameter. In this study, default parameters were used. 
 


 RESULTS

Restriction endonuclease selection
 
A total of 13 restriction enzymes were evaluated to select the most efficient and suitable restriction endonuclease to consider different parameters including average and maximum size of DNA fragment and number of fragments. The effects of different restriction endonuclease are shown in Table 1. In the case of long size fragment, KpnI enzyme showed the highest followed by NdeI and XbaI. However, none of the enzymes were found to produce more than 100 kb fragment size. Based on the rest parameters KpnI was found as the most feasible and effective restriction enzyme to have the maximum MapSets for (OM) process (Table 1).
 
Optical maps construction and assembly
 
Based on the efficiency of the enzyme, HMW DNA was digested with KpnI (New England Biolab, USA) restriction enzyme on the optical chips followed by subsequent dyeing that generated 71 GB raw data from 19 MapCards (Table 2). A total of 270,343 Single-Molecule Restriction Fragments (SMRMs) with an average size of 263.22 kb were produced from the optical chips analysis (Table 2). Within total molecules, 5,007,936 fragments were found having an average size of 14.209 kb. In addition to this, assembly of the molecules produced 12 unambiguous super-scaffolds (denoted as chromosomes) that are terminated   with   telomeric blunt ends (Figure 2).  The chromosomes ranged from 1.6 to 6.7 Mb in sizes and spanned a total 49.723 Mb by joining all SMRMs through ArgusTM optical mapping system.  It was also observed that optical mapping reduced the number of scaffolds from 94 to 88 where the largest scaffold increased by ~2 Mb in size. The indications in terms of contiguity along with quality and improvement, N50 placed on scaffold number 5 of OM instead of scaffold 6 of NGS. It increased by 4.25 Mb from 3.39 of NGS. Correspondingly, N90 also changed over its place on scaffold 11 instead of 14 by increasing size 2.9 Mb from 1.4. Although the N rate increased by 0.09%, still the GC content was unchanged (Table 3).
 
 
 
These results clearly pointed out the improvement of the assembly quality of M. phaseolina MS6 genome.
 
Alignment features between optical maps and reference maps
 
In alignment matrix, among all scaffolds only 17 were anchored on 11 chromosomes that spanned over 93.31% of the genome while none were on chromosome 12 (Figures 3 and 4). The sizes of aligned and non-aligned scaffolds were  46.35  and  3.3 Mb,  respectively.  Optical mapping deciphered 107 gaps and 4 overlaps size totaling 9.98 Mb and 26,040 kb, respectively, while 18% of the gaps can be closeable (Table 4).
 
A total number of 12 inversions (map is in reverse orientation) having 17.07 Mb in size were identified and made corrected for sequence finishing (Supplementary Files 1 and 2).
 


 DISCUSSION

We have constructed whole-genome optical maps of   M. phaseolina MS6 genome based on HMW DNA shearing by KpnI restriction endonuclease using OpGenTM optical mapping approach.. This technology is used to pick out the different types of incongruity between sequence generated in silico map and optical map along with current sequence validation of total spanned assembly. These issues were achieved by a series of action like-(i) alignment of optical map with in silico restriction map to find out all types of error, (ii) characterization of sequence contigs in respect to finding out the gaps, and (iii) the sequences were validated and placed on the optical map resulting in an explicit sequence validation. In the optical mapping   process,  restriction  endonuclease  is  used  to digest total genomic DNA as a single molecule of >200 kb  in  size  that  was  assembled  into  an  ordered  high-resolution restriction map possessing all fundamental genomic bases (Reslewic et al., 2005). This iterative  and computational assembly process joined all the SMRMs into super scaffolds by accomplishing sufficient representation across the chromosomes by coverage, the sufficient number of molecule maps covering each restriction fragment of the chromosome (depth >30X) and represents the genome complexity in terms of composition and structure (Ghurye and Pop, 2019). Finally, the optical mapping process generated 12 chromosomes terminating with both chromosomal telomeric blunt ends (blunt ends are not enzyme cut sites rather than the true end of a chromosome where the SMRMs ended at the same sequence). Furthermore, the telomeric end sequence (TTAGGG) of filamentous fungi within optical mapping organized ordered chromosomal sequence and found every chromosome possess their telomeric repetitive nucleotide sequence in between last SMRMs’s (Average length 263 kb). The whole-genome restriction map consists of the chromosomes with dispersed arranged gaps. The similar results were observed in rice (Zhou et al., 2007) where the physical map consists of 14 contigs, covering its 12 chromosomes. In Ganoderma lucidum (Chen et al., 2012), 82 scaffolds were ordered and oriented onto 13 chromosome-wide optical maps that are very similar to our optical mapping results. The finished current NGS assembly is 49.295 Mb, that is, very close to our estimated 49.723 Mb KpnI optical restriction map. The difference between the two assemblies was around ~1% which is identified as map error denoted as gaps, misassembles, and inversions. The improved and furnished assembly along with chromosome evolution mannered study of the fungus might be used for valuable resources along with fixation of control measures by biotechnological manner.


 CONCLUSION

Here we presented an improved assembly of M. phaseolina MS6 genome with chromosomal level analyses using optical mapping data was presented. In silico analyzed 12 chromosomes with congruence and discordance makes the assembly error-free. The improved non-erroneous longer scaffold based chromosomal spanned assembly might be considered as the milestone to researchers for searching precisely its pestilential tools as well as survival dimensions in diverse environmental cues. The furnished assembly can also be used for future chromosomal re-arrangements and evolution studies in other fungi along with its control measures by developing the cross-talk phenomena between host and the pathogen.


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.


 ACKNOWLEDGEMENTS

The authors thank Professor Dr. Wang Lei, Dr. Bin Liu and Dr. Yamin San of TEDA School of Biological Sciences and Biotechnology, Nankai University, China for their comprehensive help in the experiment. The research was funded by Basic and Applied Research on Jute Project of Bangladesh Jute Research Institute, Dhaka, Bangladesh.



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