SUPPLEMENTARY INFORMATION

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Table S1 / List of genes on fosmid HF70_39H11 / 2
Table S2 / List of genes on fosmid HF70_59C08 / 3
Table S3 / List of genes on fosmid HF10_3D09 / 5
Table S4 / List of genes on fosmid HF70_19B12 / 6
Figure S1 / Phylogenetic trees of preprotein translocase SecY subunit and 13 concatenated ribosomal proteins / 7
Figure S2 / Alignment of proteorhodopsins / 8
Figure S3 / Alignments of fully sequenced fosmid clones HF70_19B12 and HF10_3D09 and fosmid clone termini / 9
Figure S4 / GC content of proteorhodopsin genes from the Sargasso Sea shotgun sequence data / 10
Supplementary Table S1. List of genes on fosmid HF70_39H11 (36119 bp, 50.0 mol% GC).
Number / Start / Stop / Strand / Description / Best BLAST hit
GenBank gi / -log(e) / % identity / Taxonomic affiliation
1 / 2 / 364 / + / Similar to threonine efflux protein / 20090952 / 8 / 26 / Methanosarcina acetivorans (Euryarchaeota)
2 / 414 / 1256 / + / Proteorhodopsin / 29470240 / 66 / 50 / Uncultured bacterium
3 / 1344 / 1592 / + / Hypothetical transmembrane protein / 39594988 / 0 / 29 / Caenorhabditis briggsae (Nematoda)
4 / 1644 / 2606 / + / Hypothetical transmembrane protein / 54295326 / 4 / 24 / Legionella pneumophila (Gammaproteobacteria)
5 / 2607 / 3227 / - / Sulfite oxidase-like protein / 23126017 / 57 / 53 / Nostoc punctiforme (Cyanobacteria)
6 / 3276 / 4163 / + / ATP-NAD kinase-like protein / 50252877 / 7 / 25 / Oryza sativa (Viridiplantae)
7 / 4166 / 4630 / + / Conserved hypothetical protein / 21674231 / 40 / 56 / Chlorobaculum tepidum (Chlorobi)
8 / 4635 / 5339 / + / rRNA methylase / 51892235 / 16 / 31 / Symbiobacterium thermophilum (Actinobacteria)
9 / 5410 / 5715 / + / Hypothetical protein / 47221082 / 1 / 28 / Tetraodon nigroviridis (Metazoa)
10 / 5725 / 5961 / + / Hypothetical protein / 51894412 / 2 / 27 / Symbiobacterium thermophilum (Actinobacteria)
11 / 6009 / 6629 / + / Hypothetical protein / 57158606 / 2 / 27 / Thermococcus kodakaraensis (Euryarchaeota)
12 / 6639 / 8036 / + / Conserved hypothetical protein / 48855519 / >100 / 48 / Cytophaga hutchinsonii (Bacteroidetes)
13 / 8060 / 9037 / + / Conserved hypothetical protein / 49475041 / 29 / 29 / Bartonella henselae (Alphaproteobacteria)
14 / 9041 / 9838 / - / Conserved hypothetical transmembrane protein / 57159238 / 12 / 28 / Thermococcus kodakaraensis (Euryarchaeota)
15 / 9848 / 11068 / - / Acetyl-CoA acetyltransferase / 51891863 / 95 / 48 / Symbiobacterium thermophilum (Actinobacteria)
16 / 11237 / 23470 / + / Domain repeat protein / 42523126 / 46 / 25 / Bdellovibrio bacteriovorus (Deltaproteobacteria)
17 / 23479 / 24789 / + / Conserved hypothetical protein / 21674826 / 27 / 30 / Chlorobaculum tepidum (Chlorobi)
18 / 24790 / 25833 / - / TraB/PrgY homolog / 11497943 / 60 / 39 / Archaeoglobus fulgidus (Euryarchaeota)
19 / 25941 / 26720 / - / Putative dihydropteroate synthase / 53766060 / 57 / 47 / Rubrobacter xylanophilus (Actinobacteria)
20 / 26831 / 27538 / + / Hypothetical protein / 13488528 / 2 / 26 / Mesorhizobium loti (Alphaproteobacteria)
21 / 27543 / 28097 / + / RNA methylase-like protein / 20094240 / 37 / 44 / Methanopyrus kandleri (Euryarchaeota)
22 / 28136 / 29452 / + / Carbamoylphosphate synthase small subunit / 15669208 / 71 / 43 / Methanocaldococcus jannaschii (Euryarchaeota)
23 / 29436 / 32792 / + / Carbamoylphosphate synthase large subunit / 22095481 / >100 / 50 / Methanothermobacter thermautotrophicus (Euryarchaeota)
24 / 32793 / 34070 / - / Hypothetical protein / 48768476 / 3 / 25 / Ralstonia metallidurans (Betaproteobacteria)
25 / 34120 / 35916 / - / Lysyl-tRNA synthetase / 48853080 / 87 / 33 / Ferroplasma acidarmanus (Euryarchaeota)
26 / 35927 / 36118 / - / Orotate phosphoribosyltransferase-like protein / 48853079 / 10 / 46 / Ferroplasma acidarmanus (Euryarchaeota)
Supplementary Table S2. List of genes on fosmid HF70_59C08 (37383 bp, 51.4 mol% GC).
Number / Start / Stop / Strand / Description / Best BLAST hit
GenBank gi / -log(e) / % identity / Taxonomic affiliation
1 / 3 / 107 / + / Conserved hypothetical protein / 47079401 / 10 / 80 / DeepAnt-JyKC7 (Euryarchaeota)
2 / 110 / 970 / + / Similar to heterodisulfide reductase B subunit / 47079402 / 97 / 64 / DeepAnt-JyKC7 (Euryarchaeota)
3 / 967 / 3603 / - / ATP-dependent RNA helicase / 47079403 / >100 / 42 / DeepAnt-JyKC7 (Euryarchaeota)
4 / 3907 / 4923 / + / Ribosomal protein L3 / 47079404 / >100 / 65 / DeepAnt-JyKC7 (Euryarchaeota)
5 / 4927 / 5790 / + / Ribosomal protein L4 / 47079405 / >100 / 70 / DeepAnt-JyKC7 (Euryarchaeota)
6 / 5796 / 6083 / + / Ribosomal protein L23 / 47079406 / 34 / 75 / DeepAnt-JyKC7 (Euryarchaeota)
7 / 6098 / 6817 / + / Ribosomal protein L2 / 47079407 / >100 / 79 / DeepAnt-JyKC7 (Euryarchaeota)
8 / 6824 / 7342 / + / Ribosomal protein S19 / 47079408 / 55 / 60 / DeepAnt-JyKC7 (Euryarchaeota)
9 / 7356 / 7883 / + / Ribosomal protein L22 / 47079409 / 68 / 71 / DeepAnt-JyKC7 (Euryarchaeota)
10 / 7883 / 8689 / + / Ribosomal protein S3 / 47079410 / 91 / 64 / DeepAnt-JyKC7 (Euryarchaeota)
11 / 8689 / 8889 / + / Ribosomal protein L29 / 47079411 / 20 / 76 / DeepAnt-JyKC7 (Euryarchaeota)
12 / 8897 / 9193 / + / Translation initiation factor SUI1 / 47079412 / 45 / 88 / DeepAnt-JyKC7 (Euryarchaeota)
13 / 9197 / 9454 / + / Ribonuclease P protein component 1 / 47079413 / 15 / 52 / DeepAnt-JyKC7 (Euryarchaeota)
14 / 9460 / 9795 / + / Ribosomal protein S17 / 47079414 / 43 / 72 / DeepAnt-JyKC7 (Euryarchaeota)
15 / 9792 / 10190 / + / Ribosomal protein L14 / 47079415 / 60 / 83 / DeepAnt-JyKC7 (Euryarchaeota)
16 / 10201 / 10626 / + / Ribosomal protein L24 / 47079416 / 37 / 51 / DeepAnt-JyKC7 (Euryarchaeota)
17 / 10623 / 11321 / + / Ribosomal protein S4 / 47079417 / 79 / 62 / DeepAnt-JyKC7 (Euryarchaeota)
18 / 11339 / 11845 / + / Ribosomal protein L5 / 47079418 / 70 / 73 / DeepAnt-JyKC7 (Euryarchaeota)
19 / 11848 / 12000 / + / Ribosomal protein S14 / 47079419 / 21 / 88 / DeepAnt-JyKC7 (Euryarchaeota)
20 / 12013 / 12402 / + / Ribosomal protein S8 / 47079420 / 57 / 77 / DeepAnt-JyKC7 (Euryarchaeota)
21 / 12413 / 12961 / + / Ribosomal protein L6 / 47079421 / 70 / 67 / DeepAnt-JyKC7 (Euryarchaeota)
22 / 12974 / 13606 / + / Ribosomal protein L32 / 47079422 / 42 / 70 / DeepAnt-JyKC7 (Euryarchaeota)
23 / 13606 / 14064 / + / Ribosomal protein L19 / 47079423 / 55 / 69 / DeepAnt-JyKC7 (Euryarchaeota)
24 / 14064 / 14576 / + / Ribosomal protein L18 / 47079424 / 57 / 62 / DeepAnt-JyKC7 (Euryarchaeota)
25 / 14692 / 15345 / + / Ribosomal protein S5 / 47079425 / 78 / 74 / DeepAnt-JyKC7 (Euryarchaeota)
26 / 15405 / 15812 / + / Ribosomal protein L30 / 47079426 / 49 / 63 / DeepAnt-JyKC7 (Euryarchaeota)
27 / 15823 / 16248 / + / Ribosomal protein L15 / 47079427 / 49 / 67 / DeepAnt-JyKC7 (Euryarchaeota)
28 / 16440 / 18254 / + / Preprotein translocase subunit SecY / 47079428 / >100 / 80 / DeepAnt-JyKC7 (Euryarchaeota)
29 / 18592 / 20063 / + / 16S rRNA / 30314517 / >100 / 98 / Uncultured archaeon
30 / 20182 / 20835 / + / Similar to threonine efflux protein / 20090952 / 26 / 34 / Methanosarcina acetivorans (Euryarchaeota)
31 / 20885 / 21727 / + / Proteorhodopsin / 29470240 / 66 / 50 / Uncultured bacterium
32 / 21815 / 22072 / + / Hypothetical transmembrane protein / 46097664 / 0 / 32 / Ustilago maydis (Fungi)
33 / 22075 / 22695 / - / Sulfite oxidase-like protein / 23126017 / 56 / 52 / Nostoc punctiforme (Cyanobacteria)
34 / 22744 / 23628 / + / Similar to ATP-NAD kinase / 50252877 / 7 / 25 / Oryza sativa (Viridiplantae)
35 / 23639 / 25249 / + / Hypothetical transmembrane protein / 39593873 / 1 / 26 / Caenorhabditis briggsae (Metazoa)
36 / 25270 / 25731 / + / Conserved hypothetical protein / 21674231 / 39 / 55 / Chlorobaculum tepidum (Chlorobi)
37 / 25739 / 26443 / + / rRNA methylase / 51892235 / 16 / 31 / Symbiobacterium thermophilum (Actinobacteria)
38 / 26514 / 26819 / + / Hypothetical protein / 47221082 / 1 / 28 / Tetraodon nigroviridis (Metazoa)
39 / 26829 / 27065 / + / Hypothetical protein / 51894412 / 2 / 25 / Symbiobacterium thermophilum (Actinobacteria)
40 / 27113 / 27733 / + / Hypothetical membrane protein / 56751615 / 3 / 25 / Synechococcus elongatus (Cyanobacteria)
41 / 27743 / 29140 / + / Conserved hypothetical secreted protein / 48855519 / >100 / 48 / Cytophaga hutchinsonii (Bacteroidetes)
42 / 29164 / 30141 / + / Conserved hypothetical membrane protein / 55822830 / 28 / 29 / Streptococcus thermophilus (Firmicutes)
43 / 30145 / 30942 / - / Conserved hypothetical membrane protein / 42558685 / 13 / 27 / Arthrobacter aurescens (Actinobacteria)
44 / 30952 / 32172 / - / Acetyl-CoA acetyltransferase / 51891863 / 93 / 47 / Symbiobacterium thermophilum (Actinobacteria)
45 / 32281 / 37381 / + / Domain repeat protein / 42523126 / 45 / 24 / Bdellovibrio bacteriovorus (Deltaproteobacteria)
Supplementary Table S3. List of genes on fosmid HF10_3D09 (40468 bp, 46.8 mol% GC).
Number / Start / Stop / Strand / Description / Best BLAST hit
GenBank gi / -log(e) / % identity / Taxonomic affiliation
1 / 2 / 2156 / - / Putative alpha-agarase precursor / 6724084 / 39 / 56 / Alteromonas agarilytica (Gammaproteobacteria)
2 / 2242 / 4023 / + / ABC transporter ATP-binding protein / 22294298 / 100 / 56 / Thermosynechococcus elongatus (Cyanobacteria)
3 / 4073 / 4909 / + / Proteorhodopsin / 67906627 / 66 / 51 / Uncultured bacterium eBACmed86H08
4 / 4956 / 5912 / - / Hypothetical protein / 34497939 / 3 / 25 / Chromobacterium violaceum (Betaproteobacteria)
5 / 5970 / 6803 / - / Phosphoribosylformylglycinamidine synthase I / 71546617 / 59 / 46 / Syntrophobacter fumaroxidans (Deltaproteobacteria)
6 / 6800 / 9826 / - / Phosphoribosylformylglycinamidine synthase II / 23473641 / 100 / 49 / Desulfovibrio desulfuricans (Deltaproteobacteria)
7 / 9883 / 12816 / - / Excinuclease ATPase subunit (COG0178) / 51891305 / 100 / 54 / Symbiobacterium thermophilum (Actinobacteria)
8 / 12838 / 13722 / - / Putative RNA pseudouridylate synthase / 29337946 / 37 / 36 / Bacteroides thetaiotaomicron (Bacteroidetes)
9 / 13732 / 14769 / - / Putative molybdopterin biosynthesis MoeA protein / 46200196 / 60 / 39 / Thermus thermophilus (Deinococcus-Thermus)
10 / 15004 / 16332 / + / ABC-type sulfate/molybdate transport system, ATPase component (COG1118) / 2621548 / 39 / 44 / Methanothermobacter thermautotrophicus (Euryarchaeota)
11 / 16329 / 17123 / - / Sulfate transport system substrate-binding-like protein / 67519838 / 38 / 35 / Chromohalobacter salexigens (Gammaproteobacteria)
12 / 17498 / 18811 / - / Putative mRNA processing factor / 13541268 / 76 / 38 / Thermoplasma volcanium (Euryarchaeota)
13 / 19135 / 19347 / + / 4Fe-4S ferredoxin / 71546355 / 9 / 47 / Syntrophobacter fumaroxidans (Deltaproteobacteria)
14 / 19433 / 20044 / + / Hypothetical protein / 26190091 / 0 / 28 / Flavobacterium sp. ATCC 27551 (Bacteroidetes)
15 / 20041 / 20769 / + / Putative metal-dependent hydrolase related to alanyl-tRNA synthetase / 10175556 / 32 / 36 / Bacillus halodurans (Firmicutes)
16 / 20816 / 21517 / - / Hypothetical protein / 55741721 / 1 / 25 / Sus scrofa (Metazoa)
17 / 21736 / 22491 / + / Putative metallopeptidase / 55230052 / 23 / 37 / Haloarcula marismortui (Euryarchaeota)
18 / 22492 / 24531 / - / Hypothetical protein / 68546193 / 1 / 26 / Shewanella amazonensis (Gammaproteobacteria)
19 / 24543 / 25097 / - / Hypothetical protein / 23486696 / 9 / 43 / Plasmodium yoelii (Alveolata)
20 / 25162 / 26931 / - / Putative peptidase / 67941347 / 6 / 31 / Chlorobium phaeobacteroides (Chlorobi)
21 / 27036 / 27737 / + / Hypothetical protein / 15420806 / 0 / 25 / Macaca mulatta (Metazoa)
22 / 28053 / 28289 / - / Hypothetical protein / 23483163 / 0 / 30 / Plasmodium yoelii (Alveolata)
23 / 28565 / 29095 / - / Predicted dioxygenase of extradiol dioxygenase family (COG3565) / 67736661 / 37 / 52 / Burkholderia pseudomallei (Betaproteobacteria)
24 / 29070 / 29606 / - / Conserved hypothetical protein / 75675073 / 32 / 47 / Nitrobacter winogradskyi (Alphaproteobacteria)
25 / 29649 / 31922 / - / TPR repeat-like protein / 71673743 / 23 / 24 / Trichodesmium erythraeum (Cyanobacteria)
26 / 32096 / 34732 / + / Hypothetical protein / 74356043 / 7 / 18 / Homo sapiens (Metazoa)
27 / 34733 / 35863 / - / Proline-rich hypothetical protein / 39580550 / 15 / 37 / Caenorhabditis briggsae (Metazoa)
28 / 35904 / 37091 / - / DnaG primase-like protein / 20094055 / 72 / 38 / Methanopyrus kandleri (Euryarchaeota)
29 / 37335 / 37868 / + / Hypothetical protein / 60492746 / 2 / 30 / Bacteroides fragilis (Bacteroidetes)
30 / 37940 / 38488 / + / Hypothetical protein / 27903944 / 1 / 28 / Buchnera aphidicola (Gammaproteobacteria)
31 / 38514 / 38723 / + / Hypothetical protein / 56413459 / 1 / 37 / Salmonella enterica (Gammaproteobacteria)
32 / 38774 / 39244 / + / Hypothetical protein / 60552495 / 1 / 32 / Xenopus tropicalis (Metazoa)
33 / 39198 / 39842 / - / Phosphoribosylglycinamide formyltransferase-like protein / 15156175 / 35 / 42 / Agrobacterium tumefaciens (Alphaproteobacteria)
34 / 39940 / 40467 / - / Putative 3-isopropylmalate dehydrogenase / 19913963 / 33 / 44 / Methanosarcina acetivorans (Euryarchaeota)
Supplementary Table S4. List of genes on fosmid HF70_19B12 (37010 bp, 48.8 mol% GC).
Number / Start / Stop / Strand / Description / Best BLAST hit
GenBank gi / -log(e) / % identity / Taxonomic affiliation
1 / 1 / 2215 / - / Putative DNA deoxyribodipyrimidine photolyase / 32446346 / 94 / 32 / Rhodopirellula baltica (Planctomycetes)
2 / 2417 / 2980 / + / Uncharacterized protein with NifU-like and HesB-like domains / 67155723 / 28 / 36 / Azotobacter vinelandii (Gammaproteobacteria)
3 / 2989 / 3759 / - / Conserved hypothetical protein, DUF75 family / 57641831 / 22 / 31 / Thermococcus kodakarensis (Euryarchaeota)
4 / 3868 / 4407 / + / Hypothetical protein / 11994318 / 1 / 21 / Arabidopsis thaliana (Viridiplantae)
5 / 4404 / 4802 / + / Uncharacterized low-complexity protein (COG1357) / 67939854 / 14 / 42 / Chlorobium phaeobacteroides (Chlorobi)
6 / 4805 / 5233 / - / Predicted thioesterase (COG0824) / 66857554 / 10 / 26 / Anaeromyxobacter dehalogenans (Deltaproteobacteria)
7 / 5296 / 5802 / - / Hypothetical protein / 72160482 / 3 / 30 / Thermobifida fusca (Actinobacteria)
8 / 5945 / 8494 / + / Conserved hypothetical protein / 68547222 / 97 / 39 / Shewanella amazonensis (Gammaproteobacteria)
9 / 8575 / 8733 / + / Hypothetical protein / 21674912 / 1 / 42 / Chlorobium tepidum (Chlorobi)
10 / 8735 / 8811 / - / tRNA-Arg (anticodon GCG)
11 / 8855 / 9208 / - / Hypothetical protein / 39592380 / 0 / 31 / Caenorhabditis briggsae (Metazoa)
12 / 9285 / 10097 / + / Putative DNA polymerase type III, exonuclease domain / 68192941 / 18 / 36 / Mesorhizobium sp. BNC1 (Alphaproteobacteria)
13 / 10084 / 11292 / - / Hypothetical protein / 54025152 / 5 / 25 / Nocardia farcinica (Actinobacteria)
14 / 11309 / 12112 / + / Conserved hypothetical protein, DUF75 family / 14590070 / 19 / 30 / Pyrococcus horikoshii (Euryarchaeota)
15 / 12109 / 14232 / - / NAD-dependent DNA ligase (COG0272) / 77545890 / 100 / 35 / Pelobacter carbinolicus (Deltaproteobacteria)
16 / 14283 / 14360 / - / tRNA-Pro (anticodon GGG)
17 / 14415 / 14618 / - / DNA-directed RNA polymerase subunit N (pfam01194) / 11134415 / 17 / 56 / Pyrococcus horikoshii (Euryarchaeota)
18 / 14625 / 15062 / - / Ribosomal protein S9P (pfam00380) / 134032 / 37 / 61 / Haloarcula marismortui (Euryarchaeota)
19 / 15062 / 15547 / - / Ribosomal protein L13P (pfam00572) / 11498728 / 23 / 42 / Archaeoglobus fulgidus (Euryarchaeota)
20 / 15540 / 15905 / - / Ribosomal protein L18E (COG1727) / 15790217 / 22 / 48 / Halobacterium sp. NRC-1 (Euryarchaeota)
21 / 16014 / 23999 / - / Hypothetical protein / 68547222 / 53 / 26 / Shewanella amazonensis (Gammaproteobacteria)
22 / 24138 / 24521 / + / Putative aspartate decarboxylase / 59801048 / 36 / 61 / Neisseria gonorrhoeae (Betaproteobacteria)
23 / 24531 / 24974 / - / Uncharacterized protein, DUF163 family / 66856188 / 12 / 29 / Anaeromyxobacter dehalogenans (Deltaproteobacteria)
24 / 25024 / 25347 / + / Hypothetical protein / 47076982 / 0 / 36 / Homo sapiens (Metazoa)
25 / 25347 / 26072 / + / Hypothetical protein / 13361606 / 1 / 30 / Escherichia coli (Gammaproteobacteria)
26 / 26092 / 26709 / + / Cobalamin adenosyltransferase (pfam01923) homolog / 67930057 / 34 / 42 / Solibacter usitatus (Acidobacteria)
27 / 26706 / 27113 / - / Putative NUDIX domain protein / 18159219 / 19 / 42 / Pyrobaculum aerophilum (Crenarchaeota)
28 / 27184 / 28161 / + / Hypothetical protein / 68179138 / 8 / 31 / Desulfuromonas acetoxidans (Deltaproteobacteria)
29 / 28222 / 31527 / + / Hypothetical protein / 62321593 / 4 / 21 / Arabidopsis thaliana (Viridiplantae)
30 / 31521 / 32192 / - / SAM-dependent methyltransferase / 75910754 / 36 / 36 / Anabaena variabilis (Cyanobacteria)
31 / 32301 / 33119 / + / Proteorhodopsin / 67483476 / 67 / 55 / uncultured bacterium
32 / 33210 / 34433 / - / Isocitrate dehydrogenase (COG0538) / 74311695 / 100 / 70 / Shigella sonnei (Gammaproteobacteria)
33 / 34512 / 34901 / + / Predicted redox protein / 76794593 / 13 / 39 / Pseudoalteromonas atlantica (Gammaproteobacteria)
34 / 35055 / 37008 / + / Ribonucleotide reductase alpha subunit (COG0209) / 68191395 / 100 / 60 / Mesorhizobium sp. BNC1 (Alphaproteobacteria)

ab

Supplementary Figure S1. Phylogenetic trees.(a) Preprotein translocase SecY subunit. (b) 13 concatenated ribosomal proteins (L2P, L3P, L4P, L6P, L14P, L15, L32E, S3P, S5E, S8, S14AB, S17P, S19P). The crenarchaeal sequences were used as outgroup. The trees are based on protein sequences and neighbor-joining distance calculations. The node support is shown for neighbor-joining distance analysis and maximum-likelihood analysis.

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Supplementary Figure S2. Alignment of proteorhodopsins. Some sequences are incomplete. All of the archaeal proteorhodopsins have a glutamine (Q) residue in position 134. This suggests they absorb in the blue region of the spectrum, which has been implicated as an adaptation to the light availability in the lower photic zone (Man, D., et al. EMBO J.22, 1725-1731 (2003)).

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Supplementary Figure S3. Alignments of fully sequenced fosmid clones and fosmid clone termini.(a) HF70_19B12 and (b) HF10_3D09 (fully sequenced and shown in thick black lines). Fosmid clone termini from the genomic libraries are shown in green. Thin black lines indicate putative sequence similarity with the fully sequenced fosmids. Red indicates a proteorhodopsin gene.

Supplementary Figure S4. GC content of proteorhodopsin genes from the Sargasso Sea shotgun sequence data (Venter, J. C., et al. Science 304, 66-74 (2004)). A total of 272 gene fragments of at least 450 base pairs were analyzed.

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