Article Figures & Data
Figures
- Fig. 1.
Overview of sequencing strategies used to prepare viral genomic DNA samples for 454 sequencing.
- Fig. 2.
Linear regression analysis of the percent GC content of the largest contig versus average read length for nine unknown genomes and the mock metagenome. The percent GC content for the mock metagenome represents the collective percent GC for all nine reference genomes.
- Fig. 3.
Comparison of the predicted sequence coverage of each member of the mock metagenome to the experimental coverage (± SD). The predicted coverage was normalized to the experimental average read length and the number of sequence reads obtained after quality screening.
- Fig. 4.
Box-and-whisker plots of the sequence coverage range as a function of phage genome GC content. Phage names are provided above each panel. (a) The Roche GS FLX Titanium general library preparation method. (b) The Nextera method with high-GC-content phage. (c) The Nextera method with low-GC-content phage. Whiskers represent the 5th and 95th percentiles; open diamonds represent mean values.
- Fig. 5.
Average GC content (± SD) of regions with low or high coverage in comparisons of individual phage genomes. (a) Phage comprising the mock metagenome and unknown phage genomes prepared using the Nextera protocol. (b) Mycobacteriophage genomes prepared using the Roche GS FLX Titanium general library preparation method. Values in parentheses after the phage name indicate the average % GC content of the genome. Classification of the results into low- and high-coverage areas corresponded to the regions above or below 1.5 standard deviations from the average coverage. Genomes with at least 5 regions of low and high coverage of 50 bp or longer were included in the analysis.
Tables
- Table 1.
DNA yields and fragmentation results after completion of the Nextera protocol
Phage Typea Final DNA concn (ng/μl) Total DNA (ng)b Concn fragments of 300–800 bpc (ng/μl) % total DNA (300–800 bp)d Fragment size of greatest abundancec (bp) Avg read length for 454 sequences (bp)e Angelica M 12 307 9 79 569 373 Athena M 33 858 27 81 1,402 382 Avrafan M 21 322 11 49 843 355 Blue7 M 13 332 9 68 398 336 Wee M 19 482 12 65 529 348 TUSD 1 Cy 34 915 20 58 1,392 320 TUSD 14 Cy 21 571 11 51 1,348 307 TUSD 20 Cy 20 536 10 52 732 317 TUSD 21 Cy 29 778 11 39 1,359 315 Mock metagenome 22 708 12 55 1,151 349 - Table 2.
Results determined for de novo assemblies of 454 pyrosequencing reads from Nextera and LASL fragment libraries
Phage No. of reads No. of contigs N50 contig (bp) N90 contig (bp) Major contig(s) of alignmenta Before cleaning After cleaning Before cleaning After cleaning Size(s) (bp) No. of reads Avg coveragec (± SD) Maximum coveragec Pairwise identity (%) GC (%) Angelica 4,908 3,968 11 7 28,452 18,196 28,452 1,677 22 (12) 73 99.5 66.9 18,196 1,396 29 (15) 73 93.1 65.8 Athena 18,599 15,198 186 5 69,410 69,410 69,410 15,180 84 (39) 253 99.1 67.5 Avrafan 15,562 12,975 4 2 41,902 41,902 41,902 12,970 111 (60) 349 99.4 66.6 Blue7 32,010 25,495 115 1 52,233 52,233 52,233 25,490 165 (54) 381 99.4 61.3 Wee 18,525 16,012 12 2 59,245 59,245 59,245 16,004 95 (39) 273 99.4 61.8 TUSD 1b 13,763 9,942 272 158 37,944 378 50,727, 37,944 5,665, 3,435 36 (11), 29 (11) 84, 70 99.4, 99.4 36.7, 37.3 TUSD 14 15,345 7,568 1,139 201 497 350 38,178 6,628 55 (16) 108 99.4 37.3 TUSD 20 (Nextera) 18,066 14,311 53 31 38,098 443 38,098 14,151 118 (31) 210 99.3 37.3 TUSD 20 (LASL) 12,705 8,855 – 20 7,610 726 7,610 1,005 26 (13) 67 99.0 38.0 TUSD 21b (Nextera) 16,061 11,184 88 5 36,669, 32,801 465, 315 36,669, 37,626 9,403, 1,754 81 (27), 15 (6) 173, 35 99.1, 99.4 35.9, 37.3 TUSD 21b (LASL) 31,069 20,588 62 37,873, 32,801 12,916, 6,640 71 (188), 39 (27) 2,326, 221 99.4, 98.9 37.2, 35.7 - Table 3.
Predicted versus experimental coverage and percent abundance of reads for each phage in the mock metagenome
Phage Virus typea Genome size (bp) GC content (%) Predicted result Experimental result Recruitment to reference sequence Mock assembly No. of reads Coverage Abundance of reads (%) No. of reads Coverageb (± SD) Abundance of reads (%) No. of contigs Longest contig (bp) N50 contig (bp) T4 C 166,000 35 20,000 47 28.1 31,049 61.6 (14.6) 48.2 3 169,170 169,170 Catera M 153,766 65 14,000 36 19.7 7,592 18.0 (8.6) 11.8 4 85,298 85,298 Fruitloop M 58,471 62 12,000 82 16.8 8,745 53.3 (18.9) 13.6 1 58,308 58,308 Gumball M 64,807 60 10,000 62 14.0 9,999 56.4 (18.8) 15.5 1 64,807 64,807 Omega M 110,865 61 8,000 29 11.2 3,362 10.9 (5.2) 5.2 6 41,626 20,153 Porky M 76,312 63 4,000 21 5.6 2,038 9.7 (5.8) 3.2 7 34,434 17,944 Solon M 49,487 64 2,000 16 2.8 715 5.2 (3.4) 1.1 12 10,570 7,363 P-SS2 Cy 107,530 52 1,000 3.7 1.4 757 2.5 (2.2) 1.2 81 2,924 1,313 S-SM1 Cy 174,079 41 200 0.5 0.3 205 0.4 (0.9) 0.3 50 915 463 Total 71,200 64,718 (257 unassigned, 64,462 assigned) 172 (6 unassigned, 166 assigned)
Supplemental Material
Files in this Data Supplement:
- Supplemental Figure 1 -
Histogram of the relative frequency of fragments sizes for mycobacteriophage Athena genomic DNA (Fig. S1), linear regression
analysis of the percent GC of the largest contig versus the average read length for the nine phage that compose the mock metagenome
(Fig. S2), length distribution of assembled sequencing reads generated from the Nextera system before quality screening (Fig.
S3), identical sites and pairwise identities of mock metagenome reads which
recruited to phage T4, Catera, Gumball, and P-SS2 (Table S1), and comparison of the listed genome lengths for Athena, Avrafan,
Blue7, and Wee on the Mycobacteriophage database to the consensus sequence length of the major contig generated from de novo assembly (Table S2).
PDF file, 1.2 MB.
- Supplemental Figure 1 -
Histogram of the relative frequency of fragments sizes for mycobacteriophage Athena genomic DNA (Fig. S1), linear regression
analysis of the percent GC of the largest contig versus the average read length for the nine phage that compose the mock metagenome
(Fig. S2), length distribution of assembled sequencing reads generated from the Nextera system before quality screening (Fig.
S3), identical sites and pairwise identities of mock metagenome reads which
recruited to phage T4, Catera, Gumball, and P-SS2 (Table S1), and comparison of the listed genome lengths for Athena, Avrafan,
Blue7, and Wee on the Mycobacteriophage database to the consensus sequence length of the major contig generated from de novo assembly (Table S2).
