Bull 39 Al-Meshhdany and Hassan Bull. Iraq nat. Hist. Mus. (2020) 16 (1): 39- 61. https://doi.org/10.26842/binhm.7.2020.16.1.0039 FIVE DIATOM SPECIES IDENTIFIED BY USING POTENTIAL APPLICATION OF NEXT GENERATION DNA SEQUENCING Warqaa Yehia Al-Meshhdany* Fikrat M. Hassan** *Institute of Genetic Engineering and Biotechnology for Postgraduate Studies, University of Baghdad, Baghdad, Iraq **Department of Biology, College of Science for Women University of Baghdad, Baghdad, Iraq **corresponding author: fikrat@csw.uobaghdad.edu.iq Received Date: 28 January 2020; Accepted Date: 06 April 2020; Published Date: 24 June 2020 ABSTRACT Molecular barcoding was widely recognized as a powerful tool for the identification of organisms during the past decade; the aim of this study is to use the molecular approach to identify the diatoms by using the environmental DNA. The diatom specimens were taken from Tigris River. The environmental DNA(e DNA) extraction and analysis of sequences using the Next Generation Sequencing (NGS) method showed the highest percentage of epipelic diatom genera including Achnanthidium minutissimum (Kützing) Czarnecki, 1994 (21.1%), Cocconeis placentula Ehrenberg, 1838 ( 21.3% ) and Nitzschia palea (Kützing) W. Smith, 1856 (16.3%). Five species of diatoms: Achnanthidium minutissimum; Fistulifera saprophila (Lange- Bertalot & Bonik) Lange-Bertalot, 1997; Gomphonema pumilum (Grunow) E. Reichardt & Lange-Bertalot, 1991; Navicula veneta Kützing, 1844 and Thalassiosira pseudonana Hasle Heimdal, 1970 were registered in NCBI under the accession numbers as follows: MN749640.1, MN749641.1, MN749642.1, MN749643.1 and MN749646.1 for the first time; while the two algae Fistulifera saprophila and Thalassiosira pseudonana are regarded as a new record to algal flora in Iraq. The environmental DNA study will be a catalyst for new studies of biodiversity and environmental studies in Iraq and the region. Keywords: Algae, Diatoms, Edema, Freshwater, NGS, Tigris River. INTRODUCTION Significant environmental problems are caused by rapid population growth in the world; lack of environmental knowledge in society and changes in the industry, particularly during 40 Five diatom species identified the last century. Freshwater habitats are without doubt one of the biosphere elements most impacted by this pollution. Monitoring water quality is therefore essential to the health of the water ecosystem's sustainability and protection (Çiçek et al., 2013; Campbell et al., 2017). Monitoring of water quality by physical and chemical methods is inadequate; in the recent years, particularly in the scientific community, the biological monitoring methods and biological indicator organisms were widely used for effective research (Chang, 2008; Tokatlı and Dayıoğlu, 2011; Adebayo et al., 2013; Berthold et al., 2018). Diatoms are considered to be a large part of the benthos (often 90–95 percent), and are present all the time in all surface waters. They are also one of the most important groups of aquatic producers and react quickly to the environmental variables change. Diatoms, which are recognized as an important component of bioindicator species, have, therefore, been used as water pollution indicators for environmental assessments in many countries (Gürbüz and Kivrak, 2002; Passy et al., 2004; Godhe and Härnström, 2010; Aydın and Büyükışık, 2014; Tan et al., 2017). Recently, scientists and researchers can use a basic reality to obtain information and produce more informed choices; this material persists, giving insight into the creature's past and present that left it behind. The eDNA samples were taken from different environments and for that it called environmental DNA (Thomsen and Willerslev, 2015). During the past decade, molecular barcoding has been widely recognized as a powerful tool for identifying species. The assumption is that there is sufficient information in a short DNA sequence (DNA barcode) to identify the organisms. The major advantage in design studies of the use of DNA barcodes is that standardization and process implementation are simpler than the conventional morphology-based approach (Gao, 2019). Metabarcoding, which refers to the employment of universal primers for the amplification of DNA from various organisms collected in one sample, is the approach that is most commonly applied in Next Generation Sequencing (NGS) (Taberlet et al., 2012). NGS approaches are being increasingly employed to characterize water-living organisms from the eDNA specimens (Yu et al., 2015). Current advances in NGS approaches have made it possible to employ molecular barcoding in readily and efficiently investigating the diversity in the environment. The NGS -based environmental monitoring has been shown to be of less time and cost-consuming as compared to the conventional morphology-based methods (Baird and Hajibabaei, 2012). It is important to use the molecular concept as a solution to revise the mis-identification of diatoms and it could be also useful for biodiversity studies (Vasselon et al., 2017). The diatoms identification are often collected as a mixed species in taken sample and this is the main challenges for this purpose (Zimmerma et al., 2015). 41 Al-Meshhdany and Hassan Al-Rawi et al. (2018) reported that the traditional classification is not accurate to identify the algal species in Iraq and confirmed the use of molecular concept to identify algal taxa; Abed et al. (2018) used the molecular concept to identify the algal Coelastrella Chodat, 1922. This study is aimed to assess the suitability of amplicon sequencing in Next Generation Sequencing (NGS) approach using Illumina platform for identifying epipelic diatoms in the sediment of the Tigris River for the evaluation and development of molecular biological methods in water quality. MATERIAL AND METHODS Specimens collection Algal specimens were collected from five sites along the Tigris River from November 2018 to July 2019 (Map 1, Tab. 1); specimens of epipelic were collected randomly by scraping the clay from the surface layer with a depth 0.1-0.5 cm in area (50 m2) and (3-5 mm) using a spatula, samples were placed in polyethylene bottles and sample water was added, the bottle was closed and shaken well and placed in a dark place until returning to the laboratory. Epipelic diatoms were trapped by lens tissues as described by Eaton and Moss (Salman et al., 2017). Epipelic cell was identified after cleaning the silica skeleton by placing the glass slide on a heating plate (75-80°C), then placing a droplet of the sample on the slide and letto dry completely; followed by a concentrated nitric acid was added on the dry spot and the acid was left to evaporate completely. Then slides were mounted by Canada balsam and the cover was flipped on the dry spot and the lid of the slide was pressed gently to distribute the material in a homogeneous manner to avoid the emergence of bubbles near the edges of the sliding lid (Salman et al. 2017); diatoms were identified according to Round et al (1990). Map (1): Map of study areas (Source: https://earth.google.com). 42 Five diatom species identified Table (1): Geographical positions (GPS) of the five study sites. Culturing of diatoms Diatoms were cultured by using the purchase F2 medium following Guillard (1975(method. Each epipelic diatom cell suspension was inoculated with 20 to 250 ml off/2+0.025 SiO3 medium gradually, the culture was incubated under cycles of illumination with 12 h/12 h light-dark and constant temperatures 20°C (Al-Hussieny et al., 2014).One ml of culture was transferred to 1.5 ml tubes in the exponential growth phase (14-20 days of incubation), and the sample was centrifuged at 4000 xg for ten minutes. In the final step, the supernatant was discarded and the resulted pellets were stored at -20 °C, this step is to freeze the pellet in order to block the action of the enzymes like RNAase and protease. The pellets were kept for further use as recommended by Visco et al. (2015). Molecular identification of Diatoms In order to identify unknown diatoms at a molecular base, four genes were selected (XXXX) (Tab. 2). Primers were designed and manufactured in Macrogen company laboratories (Seoul, South Korea). Table (2): Primers design used in this study. Genomic DNA manipulation: For DNA purification, the genomic DNA of 20 isolated samples of unknown diatoms were extracted according to the protocol of QIAamp DNA Mini Kit, QIAGEN, and the isolated DNA was subjected to PCR (Gene Amp, PCR system 9700; Applied Biosystem) according to manufacturer's instructions. No. Symbol Area Coordinate North East 1 S1 Al-Muthanna Bridge 33°25'41.85″ 44°20'49.63″ 2 S2 Al-Sarafiya Bridge 33°2112.99″ 44°22'28.77″ 3 S3 Al-Shuhadaa Bridge 33°2019.99″ 44°23'19.91″ 4 S4 Al-Jadriya 33°16'58.35″ 44°22'31.87″ 5 S5 Al-Zafraniya 33°17'25.44″ 44°26'58.23″ 43 Al-Meshhdany and Hassan Multiplex polymerase chain reaction (PCR) The total volume of PCR amplification reaction was performed 25µ land included10ng/µl DNA, (1X) Taq PCR PreMix (Intron, Korea), and 1µM of each primer, and then distilled water was added into the tubes. Conditions of the thermal cycling containing denaturation at 95 °C for 5 min, were followed by 30 cycles of 95 °C for 30s, 60 °C for 30s and 72 °C for 30s, with a final incubation at 72 °C for 7 min using a thermal Cycler (Gene Amp, PCR system 9700; Applied Biosystem). The PCR products were separated by 2% agarose gel electrophoresis and visualized by exposure to ultraviolet light (302 nm) after staining with red stain (Intron Korea). For PCR products, 10μl was directly loaded into the well. Electrical power was turned on at 100v/m Amp for 75 minutes and DNA was migrating from the Cathode to plus Anode poles. Ethidium bromide-stained bands in gel were visualized using gel imaging system. For standard genes sequencing, PCR amplification of 18S rRNA products of all isolated diatoms was sent to macrogen company laboratories for sequencing using the Illumina platform by Next Generation Sequencing (NGS) workflow, which includes 4 basic steps. Calculating Phred Quality Scores (Q scores) Q scores are a measure of the quality of the identification of the nucleobase generated by automated DNA sequencing, that is logarithmically related to the base call error probabilities (P)(Ewing and Green,1998). Q = − 10 log10P RESULTS AND DISCUSSION A total of 186 epipelic diatoms taxa were identified according to the traditional concept (by compound microscopy model GX- 140105) which belong to a 59 genera according to (Round et al., 1990). The most abundant taxa are illustrated in Table (3). 44 Five diatom species identified Table (3): The most abundant diatomic taxa (identified by compound microscope) during the study period. Characterization of Diatoms by 18S rRNA and eDNA To unequivocally determine the diatoms in sediment samples, diatoms were isolated by two means through 18S rRNA and eDNA. The gene of interest was screened for 18SrRNA using different primer pairs (Table 4).The resulted PCR products of 18S rRNA were obtained from unknown samples and analyzed on 2% agarose gel and subsequently sequenced by NGS. The PCR products were (778bp) for A. minutissimum, (877 bp) for F. saprophila, (1110 bp) G. pumilum, (679bp) N. veneta, and (484 bp) for T. pseudonana (Pl. 1). Table (4): Data Statistics for diatoms. Genes Total read bases (bp) Total reads GC (%) AT (%) Q20 (%) Q30 (%) 18S_V9FV9R 94,387,580 313,580 47.957 52.04 77.987 72.456 D2D3_LSU 98,594,356 327,556 50.856 49.14 89.683 78.397 ITS3_ITS4 102,630,164 340,964 43.632 56.37 95.028 88.079 Classes Taxa Bacillariophycaeae Achnanthidium minutissimum (Kutzing) Czarnecki Cocconeis placentula Ehrenberg C. placentula var. euglypta (Ehrenberg) Grunow Gomphonema gracile Ehrenberg Nitzschia frustulum var. minuta Pantocsek Rhopalodia musculus (Kützing) O.Müller Fragilariophyceae Fallacia enigmatica (H. Germain) Lange-Bertalot & Werum Fragilaria intermedia (Grunow) Grunow F. pygmaea (Kützing) A. J. Stickle & D.G.Mann Coscinodiscophyceae Aulacoseira granulata (Ehrenberg) Simonen Melosira varians C.Agardh Pantocsekiella ocellata (Pantocsek) K.T.Kiss & E.Ács 45 Al-Meshhdany and Hassan Plate (1):PCR products were electrophoresed on a 2% agarose gel (2 h., 5V/cm, 1X TBE) and visualized under U.V. light after staining Lane: L (M: 100bp ladder, S: sample. Lane S1, S2, S3, S4 and S5 represent the PCR products of isolated diatoms. In Illumina MiSeq by NGS, the sequencing generated total number of bases sequenced, and total number of reads sequences, quinine- cytosine (GC %) content and adenine - thymine (AT%). As is explained table (4). While the high quality of the phred score for each gene sequences with an average Q20% and Q30% was illustrated in Diagram (1). Diagram (1): Quality values line about sequences of the three genes with Q20/Q30 scores of sequences data. The following diatom species were obtained with the relative abundance by the laboratories of Macrogen Corporation laboratories in Korea using Illumina platform by NGS for each DNA samples. These samples were identified by a three encoding genomic sequence described in previous table 2. A. minutissimum and C. placentula were diagnosed with the highest relative abundance with a slight difference (21.1and 21.3%), respectively, followed by N. palea with a percentage (16.3%), while the least abundance diatom was recorded for N. cf. frustulum with abundance of (0.7 %) (Tab. 5). 46 Five diatom species identified Table (5): Relative abundance of diatom species by NGS Taxa Proportion (%) Notes Achnanthidium minutissimum 21.1 For the first time identified by molecular analysis in Iraq Amphora montana 1.6 Cocconeis placentula 21.3 Cyclotella meneghiniana 2.3 Fistulifera saprophila 1.9 New record in Iraq Fragilaria pinnata 2.9 Gomphonema parvulum 9.8 Gomphonema pumilum 9.6 For the first time identified by molecular analysis in Iraq Nitzschia amphibia 1.4 Nitzschia cf. frustulum 0.7 Nitzschia palea 16.3 Navicula veneta 3.4 For the first time identified by molecular analysis in Iraq Thalassiosira pseudonana 4.34 Unclear. Thalassiosira pseudonana is considered widespread. It is known from freshwater habitats (Kiss, 1984). New record in Iraq Confirm by Prof. Dr.Bahram K. Maulood (personal communication, March 14, 2020) Ulnaria ulna 3.36 The NGS sequencing were aligned online using Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI). The 18S rRNA sequence of all diagnostic diatom samples showed 99% homology with other global diatoms registered in the NCBI under the accession numberin NCBIMN602030.1, MH997844.1, AM501970.1, KU900218.1, KC736629.1, respectively. The sequence analysis, types of polymorphism, location of nucleotide of 18S rRNA gene for isolated diatoms were shown in Table (6) and demonstrated in (Diags. 2, 6). 47 Al-Meshhdany and Hassan Table (6): Types of polymorphism of 18S rRNA gene from isolated diatoms. No. of sample Type of substitution Location Nucleotide Sequence ID Score Identities Taxa 1 Transition 762 G>A ID:MN602030.1 1372 99% Achnanthidium minutissimum Transition 977 A>G Transvertion 1211 C>G Transition 1231 G>A Transition 1250 A>G Transvertion 1354 T>G Transition 1365 A>G 2 Transition 554 A>G ID:H997844.1 1564 99% Fistulifera saprophila Transvertion 556 T>A Transition 671 A>G Transvertion 886 C>A 3 Transvertion 556 T>G ID:M501970.1 1198 99% Navicula veneta Transvertion 711 T>G Transvertion 735 T>G Transition 765 A>G Transition 792 T>C Transvertion 849 G>C 4 Transition 785 A>G ID:KU900218.1 869 99% Thalassiosira pseudonana 5 Transvertion 711 G>C ID:KC736629.1 1994 99% Gomphonema pumilum Transvertion 923 G>C 48 Five diatom species identified Achnanthidium minutissimum strain )18S rRNA) gene, partial sequence. Sequence ID:MN602030.1 Length: 1651Number of Matches: 1 Range 1: 644 to 1421Genbank Graphics Next Match Previous Match Score Expect Identities Gaps Strand 1372 bits(1521) 0.0 771/778(99%) 0/778(0%) Plus/Plus Query61GTTCAAAGCAGGCTTATGCCGTTGAATGTCTTAGCATGGAATAATAAGAT AGGACCTTAG120 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct704 GTTCAAAGCAGGCTTATGCCGTTGAATGTCTTAGCATGGAATAATAAGATAGGAC CTTGG763 Query301 CCATCGTAGTCTTAACCATAAACTATGCCGACAGGGGATTGGTGGGGTTTCGTTA CGTCT360 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct944 CCATCGTAGTCTTAACCATAAACTATGCCGACAAGGGATTGGTGGGGTTTCGTTA CGTCT1003 Query541 TCTTTCTTGATTCTATGGGTGGTGGTGGATGGCCGTTCTTAGTTGGTAGAGTGATT TGTC600 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct1184TCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGA GTGATTTGTC1243 Query601 TGGTTAGTTCCGTTAACGAACGAGACCGCTGCCTGCTAAATAGTCCAGTGAGTGA ATTTC660 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct1244TGGTTAATTCCGTTAACGAACGAGACCGCTGCCTGCTAAATAGTCCAGT GAGTGAATTTC1303 Query661 ACTGACGAGGACTTCTTAGAGGGACGTGCGTTCTATTAGACGCAGGAAGAGAGC GGCAAT720 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct1304ACTGACGAGGACTTCTTAGAGGGACGTGCGTTCTATTAGACGCAGGAAG ATAGCGGCAAT1363 Query721 AGCAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTGATGC ATT778 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 49 Al-Meshhdany and Hassan Sbjct1364AACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACAC TGATGCATT1421 Diagram (2): Sequences analysis of 18S rRNA gene for Achnanthidium minutissimum. Fistulifera saprophila isolate HYU-D033 small subunit ribosomal RNA gene, partial sequence, Sequence ID: MH997844.1 Length: 1654Number of Matches: 1 Range 1: 305 to 1181Genbank Graphics Next MatchPrevious Match Score Expect Identities Gaps Strand 1564 bits(1734) 0.0 873/877(99%) 0/877(0%) Plus/Plus Query241 CGTAGTTGGGTATGTGGTGTGCGTTGCGGCGTCCATTTGTTTGGTTCTGCCGTGAC CGCG300 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct545 CGTAGTTGGATTTGTGGTGTGCGTTGCGGCGTCCATTTGTTTGGTTCTGCCGTGAC CGCG604 Query361 CTGTGAGAAAATTAGAGTGTTCAAAGCAGGCTTATGCCGTTGAATATATTAGCAT GGAAT420 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct665 CTGTGAAAAAATTAGAGTGTTCAAAGCAGGCTTATGCCGTTGAATATATTAGCAT GGAAT724 Query541 GAACTACTGCGAAAGCATTTACCAAGGATGTTTTCATTAATAAAGAACGAAAGTT AGGGG600 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct845 GAACTACTGCGAAAGCATTTACCAAGGATGTTTTCATTAATCAAGAACGAAAGTT AGGGG904 Diagram (3): Sequences analysis of 18S rRNA gene for Fistulifera saprophila Naviculaveneta18S rRNA gene, strain AT-108Gel01 Sequence ID: AM501970.1 Length: 1745Number of Matches: 1 Range 1: 492 to 1170GenBank Graphics Next Match Previous Match Score Expect Identities Gaps Strand 1198 bits(1328) 0.0 673/679(99%) 0/679(0%) Plus/Plus Query61CAGCGCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTT GGATTTGTGG120 50 Five diatom species identified ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct552 CAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGGATT TGTGG611 Query181 AACCTGTGTGGCATTAGGTTGTCGTGCAGGGGATGCCCAGCGTTTACTGTGAAAA AATTA240 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct672 AACCTGTGTGGCATTAGGTTGTCGTGCAGGGGATGCCCATCGTTTACTGTGAAAA AATTA731 Query241 GAGGGTTCAAAGCAGGCTTATGCCGTTGAATATGTTAGCATGGAATAATGAGATA GGACT300 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct732 GAGTGTTCAAAGCAGGCTTATGCCGTTGAATATATTAGCATGGAATAATGAGATA GGACT791 Query301 CTTTCGCTATTTTGTTGGTTTGCGCGAGAAGGTAATGATTAATAGGGACAGTTGG GGCTA360 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct792 TTTTCGCTATTTTGTTGGTTTGCGCGAGAAGGTAATGATTAATAGGGACAGTTGG GGGTA851 Diagram (4): Sequences analysis of 18S rRNA gene for Navicula veneta. Thalassiosira pseudonana strain CCAP 1085/12 18S ribosomal RNA gene, partial sequence, Sequence ID: KU900218.1 Length: 1755Number of Matches: 1 Range 1: 615 to 1098GenBankGraphics Next Match Previous Match Score Expect Identities Gaps Strand 869 bits(963) 0.0 483/484(99%) 0/484(0%) Plus/Plus Query121 GGGATACCCATCGTTTACTGTGAAAAAATTAGAGTGTTTAAAGCAGGCTTGTGCC GTTGA180 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 51 Al-Meshhdany and Hassan Sbjct735 GGGATACCCATCGTTTACTGTGAAAAAATTAGAGTGTTTAAAGCAGGCTTATGCC GTTGA794 Diagram (5): Sequences analysis of 18S rRNA gene for Thalassiosira pseudonana. Gomphonema pumilum clone TCC536 18S ribosomal RNA gene, partial sequence Sequence ID: KC736629.1 Length: 1683Number of Matches: 1 Range 1: 255 to 1364GenBank Graphics Next Match Previous Match Score Expect Identities Gaps Strand 1994 bits(2210) 0.0 1108/1110(99%) 0/1110(0%) Plus/Plus Query421 ACGTTTACTGTGAAAAAATCAGCGCGTTCAAAGCAACCTTATGCTGTGAATGTAT TAGCA480 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct675 ACGTTTACTGTGAAAAAATCAGCGCGTTCAAAGCAAGCTTATGCTGTGAATGTAT TAGCA734 Query661 TAGGGGATCCAAGATGATTAGATACCATCGTAGTCTTAACCATAAACTATGCCGA CAAGG720 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct915 TAGGGGATCGAAGATGATTAGATACCATCGTAGTCTTAACCATAAACTATGCCGA CAAGG974 Diagram (6): Sequences analysis of 18S rRNA gene for Gomphonema pumilum. NGS data analysis The results were analyzed using genius software. Sequencing of genes was performed by the Seoul National Instrumentation Center for Environmental Management (SNU NICEM) online at: http:/www.mbio.ncsu.edu/bioedit/bioedit.html, using a DNA sequencer 3730XL by Applied Biosystem. A homology search was conducted using Basic Local Alignment Search Tool (BLAST) program which is available at the National Center Biotechnology Information (NCBI) online at http://www.ncbi.nlm.nih.gov and software BioEditPro. Version: 7.0.0 program. An expected value is defined to give an estimation of the number of times expected to get the same similarity coincidental and the lower the value of expecting. This indicates that the degree of similarity was high between sequences which give greater confidence; a value close to zero means that these sequences are identical and the Bit Score, which is a statistical measure of the sequence similarity and the higher value indicates a high degree of similarity. Isolated diatom samples were confirmed by sequence-based phylogenetic tree (aligned sequences were conducted using MEGA 6 program) structuring analysis using 18S ribosomal RNA (18SrRNA) gene sequencing in Diagrams (7-11). 52 Five diatom species identified Diagram (7): Phylogenetic tree of Achnanthidium minutissimum based on 18S rRNA gene sequences conferred by GeneBank data base, were analyzed and aligned through BLAST from NCBI using the Neighbor-Joining Analyses of 778bp of corresponding position of 18S rRNA gene sequence. MEGA 6 program was used for phylogenetic tree. Diagram (8): Phylogenetic tree of Fistulifera saprophila based on 18S rRNA gene sequences conferred by GeneBank data base, were analyzed and aligned through BLAST from NCBI using the Neighbor-Joining Analyses of 877 bp of corresponding position of 18S rRNA gene sequence. MEGA 6 program was used for phylogenetic tree. 53 Al-Meshhdany and Hassan Diagram (9):Phylogenetic tree of Gomphonema pumilum based on 18S rRNA gene sequences conferred by GeneBank data base, were analyzed and aligned through BLAST from NCBI using the Neighbor-Joining Analyses of 1110 bp of corresponding position of 18S rRNA gene sequence. MEGA 6 program was used for phylogenetic tree. Diagram (10): Phylogenetic tree of Naviculaveneta based on 18S rRNA gene sequences conferred by GeneBank data base, were analyzed and aligned through BLAST from NCBI using the Neighbor-Joining Analyses of 679 bp of corresponding position of 18S rRNA gene sequence. MEGA 6 program was used for phylogenetic tree. 54 Five diatom species identified Diagram (11): Phylogenetic tree of Thalassiosira pseudonana based on 18S rRNA gene sequences conferred by GeneBank data base, aligned together with yeast, were analyzed and aligned through BLAST from NCBI using the Neighbor-Joining Analyses of 484 bp of corresponding position of 18S rRNA gene sequence. MEGA 6 program was used for phylogenetic tree. The molecular analysis revealed five diatom species which were identified for the first time by molecular analysis, while two species were recorded as new species of Iraqi algal flora and were registered in NCBI under the accession number as follows: (1) Achnanthidium minutissimum (accession numberMN749640.1). (2) Fistulifera saprophila (accession number MN749641.1) new record. (3) Gomphonema pumilum (accession number MN749642.1). (4) Navicula veneta (accession number MN749643.1). (5) Thalassiosira pseudonana (accession number MN749646.1) new record. By comparing Phylogenetic tree of A. minutissimum with neighboring countries, it was 99% closer to China. When compared, Phylogenetic tree of F. saprophila turned out to be more similar 99% to the ID number diagnosed in Korea. The Phylogenetic tree for the species G. pumilum was more closely related to the registration number that was diagnosed in France as 99%.The affinity ratio was 99% phylogenetic tree of N. veneta with registration number ID: AM501970.1 which registered in Germany. Phylogenetic tree of T. pseudonana based on 18S rRNA gene sequences conferred by GeneBank data base, were analyzed and aligned through 55 Al-Meshhdany and Hassan BLAST from NCBI using the Neighbor-Joining Analyses and was also 99% recorded in the USA. The morphological and molecular (phylogenetic) determination of diatomic organisms is another potential conflict source. Firstly, there is a range of genetically distinctive forms that reflect almost all morphospecies. Secondly, some species have their own auto-ecological values subdivided into subspecies or morphological varieties. In the first case, a significant benefit for biomonitoring may be the cryptic diversity, especially when cryptic species relate to certain specific ecological conditions. The second case is more troubling because the sub- specific taxa are generally not genetically characterized (Visco et al., 2015). The quantitative analysis of NGS data gives the greatest challenge in efforts to alleviate biases in the calculation of diatom indices. In fact, numerous NGS environmental studies display contradiction between the number of sequences assigned to a given species and the number of specimens of the same species in microscopic preparations (Gibson et al., 2014) or even microbially diverse communities (Amend et al., 2010).This unbalance correlation between the multiple reading and individuals could be interpreted either by technical biases introduced during DNA extraction, PCR amplification or sequencing or by biological factors such as the variations of rRNA gene copies (Weber and Pawlowski, 2013; Pawlowski et al., 2014), which may depend on number of nuclei in genome size, or variety in size of cell (Prokopowich et al., 2003; Heyse et al., 2010). The results given in this experience study will need validation by more NGS-based surveys of diatom diversity. Indeed, substantial efforts must be done by diatom taxonomists and biologists to complete the DNA barcoding reference database and to determine the rate of genetic and morphological differences in diatom species. A total of 186 taxa were identified of epipelic algae by microscopy (Tab. 3), whereas only a few of identified epipelic (5.4%) were observed by using molecular analysis in this study (Tab. 5). While Amphora montana, Fistulifera saprophila, Nitzschia cf. frustulum and Thalassiosira pseudonana were detected by molecular analysis and not identified by microscopy. Another study also observed only 19% of identified diatoms by using molecular analysis while they identified 63 taxa by microscopy (Vasselon et al., 2017). Vasselon et al. (2017) mentioned that about 68% of diatom species identified by microscopy were incomplete in the reference database; moreover, it is important to use suitable DNA extraction methods. This finding will encourage the researcher to use the molecular analysis for identifying algae in the environment. These diatoms were found in freshwater habitats and reorganized in different regions worldwide (Reichardit, 1997; Wojtal, 2003, Novais et al., 2015). CONCLUSION The use of molecular concept of classification is important to re-check the list of the algal flora in Iraq to confirm or to amend them. The application of eDNA revealed five diatom species were a new record species of Iraqi algal flora and it will be a catalyst for new studies 56 Five diatom species identified of biodiversity and environmental studies in Iraq and the region. The molecular application will resolve the misclassification and the persistent problems of misidentification of algae. Moreover, the NGS will decrease the period of the specimen process with using automation of the protocols of molecular works and led to the increase in the number of sampling, in addition to reduce the cost of this tech. 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(2020) 16 (1): 39-61. دايتومية باستعمال التطبيقات الممكنة لتسلسل الحمض تشخيص خمسة انواع النووي البيئي من الجيل التالي حسن** فكرت مجيد يحيى المشهداني* و ورقاء ،غدادجامعةب ،التقنيات االحيائية للدراسات العليا و *معهدالهندسية الوراثية العراق، بغداد العراق، بغداد ، جامعةبغداد ،لبناتكليةالعلوم ل ،**قسم علوم الحياة 24/06/2020، تأريخ النشر: 27/04/2020، تأريخ القبول: 16/01/2020تأريخ االستالم: الخالصة الل خعرفت تشفير الباركود بشكل واسع كأداة قوية لتحديد الكائنات الحية حديد يئي لتهدفت الدراسة الحالية الستخدام المفهوم الجز لذلكالعقد الماضي. الدايتومات باستخدام الحمض النووي البيئي. ليل اخذت العينات الدايتومات من نهر دجلة، اذ بينت نتائج استخالص وتح ( NGاستخدام تسلسل الجيل التالي ) تسلسل الحمض النووي البيئي من خالل بان اعلى نسبة سجلت لكل من الدايتومات التالية : (21.1%) Achnanthidium minutissimum (Kützing) Czarnecki, 1994 وCocconeis placentula Ehrenberg, ,Nitzschia palea (Kützing) W. Smith %( و21.3)1838 . %16.3 بنسبة 1856 مرةفي المركز الوطني لمعلومات خمسة اجناس للدايتومات الول سُجلت و MN749640.1تحت ارقام االنضمام ( NCBIلتكنولوجيا الحيوية ) MN749641.1 وMN749642.1 وMN749643.1 و MN749646.1:على التوالي و هي Achnanthidium minutissim وFistulifera saprophila (Lange-Bertalot & Bonik) Lange-Bertalot, 1997 وGomphonema pumilum (Grunow) E. Reichardt & Lange-Bertalot, 1991 وNavicula 61 Al-Meshhdany and Hassan veneta Kütz. 1844 وThalassiosira pseudonana Hasle Heimdal, 1970 النوعان ؛ كما يعدFistulifera saprophila جديدا للفلورا الطحلبية في تسجيالThalassiosira pseudonana و .العراق سة الحمض النووي البيئي عامال مساعدا في اجراء دراسات جديدة تعتبر درا حول التنوع البيولوجي والدراسات البيئة في العراق والمنطقة.