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NOVOPlasty - The organelle assembler and heteroplasmy caller
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Heteroplasmy and mitogenome assembly questions #152

Closed brenna-levine closed 3 years ago

brenna-levine commented 4 years ago

Hi,

First, thanks for a great program. I've been having a lot of fun exploring our data with it.

A little background - I am working on assembling mitogenomes for a species for which I have a reference genome, using paired-end 250bp WGS data. So far, I have run NOVOplasty for about 20 samples from different populations, and all of these analyses have returned multiple contigs per sample. The lengths of the Merged contigs that are returned all exceed the known size of this genome by a few thousand base pairs (the mitogenome is ~15000bp, but the merged options are around ~18,000-20,000bp). Importantly, for most samples, one consistent contig is returned that is around 12,000 bp. Of note, we have some suspicion based off of lab work that we might be dealing with a linear or fragmented mitogenome for this species.

I have 2 questions for you:

(1) If we fail to get single circular contigs or if we continue to get merged contig arrangements that far exceed the known size of the mitogenome, do these results support our in vivo evidence of linear/fragmented mitogenome arrangements?

(2) We are very interested in using NOVOplasty to detect heteroplasmy in our mitogenomes, a phenomenon which we know to occur in our study species. Can we use just the 12,000bp contig that is assembled for most individuals as the reference and seed for heteroplasmy analyses, even though we know this leaves about 3000bp out of our genome? In other words, is it a valid method to just look for heteroplasmy in this large contig that we feel confident about?

Please let me know if any additional information would be helpful. I look forward to any advice you might provide.

Thanks,

Brenna Levine University of Tulsa

brenna-levine commented 3 years ago

Hi Nicolas,

I'm wondering if you have received this and might be able to provide some advice? Thanks!

Brenna

ndierckx commented 3 years ago

Hi Brenna,

Sorry I was really busy lately, did n't had the time to answer this.

(1) If it is linear the assembly should stop at the ends, unless there is a repetitive region at the end. If you want you can send me the extended log of a run I can have a look at it. Just put extended log to 1 in the config file

(2) Yes it is definitely the best way to do it, problematic regions or repetitive regions should be excluded from the heteroplasmy analysis. Do you have sufficient coverage? And you always ask me for advice on that because some experience in interpreting the results can be useful.

Greets,

Nicolas

brenna-levine commented 3 years ago

Hi Nicolas,

I really appreciate your help! I have attached the extended log files for 3 of my samples. Would you mind taking a look with both of my questions in mind? Could you also provide guidance regarding where to look for coverage information in the extended log?

Thanks again,

Brenna

On Mon, Oct 26, 2020 at 10:20 AM Nicolas Dierckxsens < notifications@github.com> wrote:

Hi Brenna,

Sorry I was really busy lately, did n't had the time to answer this.

(1) If it is linear the assembly should stop at the ends, unless there is a repetitive region at the end. If you want you can send me the extended log of a run I can have a look at it. Just put extended log to 1 in the config file

(2) Yes it is definitely the best way to do it, problematic regions or repetitive regions should be excluded from the heteroplasmy analysis. Do you have sufficient coverage? And you always ask me for advice on that because some experience in interpreting the results can be useful.

Greets,

Nicolas

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub https://github.com/ndierckx/NOVOPlasty/issues/152#issuecomment-716618045, or unsubscribe https://github.com/notifications/unsubscribe-auth/APGMPM7SX4474RHBI25AUY3SMWHTVANCNFSM4STY3FPA .

-- Brenna Levine, Ph.D. Postdoctoral Research Associate University of Tulsa

Project:

Project name = bed_bug Type = mito Genome range = 11000-22000 K-mer = 33 Max memory = Extended log = 1 Save assembled reads = Seed Input = bed_bug_seed.fasta Extend seed directly = Reference sequence = Variance detection = Chloroplast sequence =

Dataset 1:

Read Length = 250 Insert size = 500 Platform = illumina Single/Paired = PE Combined reads = Forward reads = reads_1.fastq.gz Reverse reads = reads_2.fastq.gz

Heteroplasmy:

Heteroplasmy = HP exclude list = PCR-free =

Optional:

Insert size auto = yes Use Quality Scores =

TAATGAATATATCAACACGTAAAACTAACCCTTTAATTAAAACATTAAATAATTTACTTATTGATCTCCCATGTCCTACAAGAATCTCCAATTGATGAAATTTTGGATCCTTACTAAGAATATGTTTGTTAATCCAATTATTAACAGGAATCTTTTTAGCCATACATTATACAGCTAACATTGAATTAGCCTTCAACAGTGTAATTCACATTATACGTAATGTAAATAATGGTTGAATAATACGAAGTAT TAATGAATATATCAACACGTAAAACTAACCCTTTAATTAAAACATTAAATAATTTACTTATTGATCTCCCATGTCCTACAAGAATCTCCAATTGATGAAATTTTGGATCCTTACTAAGAATATGTTTGTTAATCCAATTATTAACAGGAATCTTTTTAGCCATACATTATACAGCTAACATTGAATTAGCCTTCAACAGTGTAATTCACATTATACGTAATGTAAATAATGGTTGAATAATACGAAGTAT CORRECTED READ

Initial read retrieved successfully1: TAATGAATATATCAACACGTAAAACTAACCCTTTAATTAAAACATTAAATAATTTACTTATTGATCTCCCATGTCCTACAAGAATCTCCAATTGATGAAATTTTGGATCCTTACTAAGAATATGTTTGTTAATCCAATTATTAACAGGAATCTTTTTAGCCATACATTATACAGCTAACATTGAATTAGCCTTCAACAGTGTAATTCACATTATACGTAATGTAAATAATGGTTGAATAATACGAAGTAT

1

1811841 SEED_exists

250 READ_LENGTH 500 INSERT_SIZE

250 POSITION 0 POSITION_BACK LAST_CHANCE TAATGTAAATAATGGTTGAATAATACGAAGTAT READ_END TAATGAATATATCAACACGTAAAACTAACCCTT READ_START 223 MATCH_ARRAY_READ 0 MATCH_ARRAY_BACK_READ 4 TIME1

192 READ_COUNT 0 READ_EX 191 EXTENSIONS 191 AVERAGE_COVERAGE ACATGCTAACGGCGCCTCATTCTTTTTTATTTGTATATATATACATGTAGGACGAGGAATTTACTATAATTCTTATCAACTAACTAACACTTGAA BEST_EXTENSION

stop NOBACK yes LASTCHANCE 1 COUNTSEED FINISH2

2

1811841 SEED_exists

345 READ_LENGTH 500 INSERT_SIZE

345 POSITION 0 POSITION_BACK LAST_CHANCE ACTATAATTCTTATCAACTAACTAACACTTGAA READ_END TAATGAATATATCAACACGTAAAACTAACCCTT READ_START 218 MATCH_ARRAY_READ 0 MATCH_ARRAY_BACK_READ 4 TIME1

185 READ_COUNT 0 READ_EX 182 EXTENSIONS 186 AVERAGE_COVERAGE TAGTAGGAGTAATAATGTTATTACTAACAATAGCAACAGCCTTCCTAGGTTATGTTTTACCATGAGGACAAATGTCCCTGTGGGGGGCAACAGT BEST_EXTENSION

stop NOBACK yes LASTCHANCE 1 COUNTSEED FINISH2

3

1811841 SEED_exists

439 READ_LENGTH 500 INSERT_SIZE

439 POSITION 0 POSITION_BACK LAST_CHANCE ATGAGGACAAATGTCCCTGTGGGGGGCAACAGT READ_END TAATGAATATATCAACACGTAAAACTAACCCTT READ_START 214 MATCH_ARRAY_READ 0 MATCH_ARRAY_BACK_READ 4 TIME1

175 READ_COUNT 0 READ_EX 172 EXTENSIONS 182 AVERAGE_COVERAGE AATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTT BEST_EXTENSION

stop NOBACK yes LASTCHANCE 1 COUNTSEED FINISH2

4

1811841 SEED_exists

541 READ_LENGTH 500 INSERT_SIZE

541 POSITION 0 POSITION_BACK TTCTATTGAAAATGCCACATTAACACGATTTTT READ_END TAATGAATATATCAACACGTAAAACTAACCCTT READ_START 94 MATCH_ARRAY_READ 97 MATCH_ARRAY_BACK_READ

80 READ_COUNT 80 READ_EX 0 EXTENSIONS 136 AVERAGE_COVERAGE BEST_EXTENSION

OPTION1

79 READ_COUNT_BACK 79 READ_EX_BACK 0 EXTENSIONS_BACK BEST_EXTENSION_BACK

2B 1 COUNTSEED FINISH2

5

1811841 SEED_exists

541 READ_LENGTH 500 INSERT_SIZE

541 POSITION 0 POSITION_BACK USE_REGEX USE_REGEX_BACK2 TTCTATTGAAAATGCCACATTAACACGATTTTT READ_END TAATGAATATATCAACACGTAAAACTAACCCTT READ_START 102 MATCH_ARRAY_READ 110 MATCH_ARRAY_BACK_READ yes USE_REGEX_REVERSE 4 TIME1

90 READ_COUNT 89 READ_EX 0 EXTENSIONS 109 AVERAGE_COVERAGE BEST_EXTENSION

OPTION3 USE_REGEX_BACK_REVERSE

94 READ_COUNT_BACK 94 READ_EX_BACK 0 EXTENSIONS_BACK BEST_EXTENSION_BACK

5B yes REGEX_BACK 1 COUNTSEED FINISH2

6

1811841 SEED_exists

541 READ_LENGTH 500 INSERT_SIZE

541 POSITION 0 POSITION_BACK LAST_CHANCE_BACK SNP_ACTIVE USE_REGEX TTCTATTGAAAATGCCACATTAACACGATTTTT READ_END TAATGAATATATCAACACGTAAAACTAACCCTT READ_START 102 MATCH_ARRAY_READ 220 MATCH_ARRAY_BACK_READ yes USE_REGEX_REVERSE 3 TIME1

90 READ_COUNT 89 READ_EX 0 EXTENSIONS 91 AVERAGE_COVERAGE BEST_EXTENSION

OPTION3b

186 READ_COUNT_BACK 0 READ_EX_BACK 185 EXTENSIONS_BACK AAACAGTACCTTTAATTATAATAATAGTTTTATATTTATTCTTCACTATAATTGTTATTTCATCAATTGTAAATATTCATGAAGGGCCAATACGCTCGAAAAGA BEST_EXTENSION_BACK

yes LASTCHANCE_BACK 1 COUNTSEED FINISH2

7

1811841 SEED_exists

645 READ_LENGTH 500 INSERT_SIZE

541 POSITION 104 POSITION_BACK LAST_CHANCE USE_REGEX TTCTATTGAAAATGCCACATTAACACGATTTTT READ_END AAACAGTACCTTTAATTATAATAATAGTTTTAT READ_START 237 MATCH_ARRAY_READ 117 MATCH_ARRAY_BACK_READ yes USE_REGEX_REVERSE 3 TIME1

203 READ_COUNT 0 READ_EX 201 EXTENSIONS 107 AVERAGE_COVERAGE CGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATAC BEST_EXTENSION

101 READ_COUNT_BACK 101 READ_EX_BACK 0 EXTENSIONS_BACK BEST_EXTENSION_BACK

2B yes LASTCHANCE 1 COUNTSEED FINISH2

8

1811841 SEED_exists

753 READ_LENGTH 500 INSERT_SIZE

649 POSITION 104 POSITION_BACK USE_REGEX_BACK2 CTCTAATAACCCGCTAGGGGTAGATAGTAATAC READ_END AAACAGTACCTTTAATTATAATAATAGTTTTAT READ_START 99 MATCH_ARRAY_READ 130 MATCH_ARRAY_BACK_READ

83 READ_COUNT 81 READ_EX 0 EXTENSIONS 93 AVERAGE_COVERAGE BEST_EXTENSION

OPTION1 USE_REGEX_BACK_REVERSE

114 READ_COUNT_BACK 114 READ_EX_BACK 0 EXTENSIONS_BACK BEST_EXTENSION_BACK

5B yes REGEX_BACK 1 COUNTSEED FINISH2

9

1811841 SEED_exists

753 READ_LENGTH 500 INSERT_SIZE

649 POSITION 104 POSITION_BACK LAST_CHANCE_BACK USE_REGEX CTCTAATAACCCGCTAGGGGTAGATAGTAATAC READ_END AAACAGTACCTTTAATTATAATAATAGTTTTAT READ_START 106 MATCH_ARRAY_READ 249 MATCH_ARRAY_BACK_READ yes USE_REGEX_REVERSE 3 TIME1

89 READ_COUNT 87 READ_EX 0 EXTENSIONS 83 AVERAGE_COVERAGE BEST_EXTENSION

OPTION3

216 READ_COUNT_BACK 0 READ_EX_BACK 216 EXTENSIONS_BACK CCACTTCTCATAATAAAACAAGACTCAATTATATACAATTTACATAATTATAAAACTAATTCCACAATAAAATATGATATTTCCACCTCCTTAAGAAAGCTGTTTTCATCAG BEST_EXTENSION_BACK

yes LASTCHANCE_BACK 1 COUNTSEED FINISH2

10

1811841 SEED_exists

865 READ_LENGTH 500 INSERT_SIZE

649 POSITION 216 POSITION_BACK SNP_ACTIVE USE_REGEX CTCTAATAACCCGCTAGGGGTAGATAGTAATAC READ_END CCACTTCTCATAATAAAACAAGACTCAATTATA READ_START 106 MATCH_ARRAY_READ 123 MATCH_ARRAY_BACK_READ yes USE_REGEX_REVERSE

89 READ_COUNT 87 READ_EX 0 EXTENSIONS 75 AVERAGE_COVERAGE BEST_EXTENSION

OPTION3b

108 READ_COUNT_BACK 108 READ_EX_BACK 0 EXTENSIONS_BACK BEST_EXTENSION_BACK

2B 1 COUNTSEED FINISH2

11

1811841 SEED_exists

865 READ_LENGTH 500 INSERT_SIZE

649 POSITION 216 POSITION_BACK LAST_CHANCE USE_REGEX USE_REGEX_BACK2 CTCTAATAACCCGCTAGGGGTAGATAGTAATAC READ_END CCACTTCTCATAATAAAACAAGACTCAATTATA READ_START 229 MATCH_ARRAY_READ 133 MATCH_ARRAY_BACK_READ yes USE_REGEX_REVERSE 6 TIME1

197 READ_COUNT 0 READ_EX 195 EXTENSIONS 86 AVERAGE_COVERAGE TGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT BEST_EXTENSION

USE_REGEX_BACK_REVERSE

122 READ_COUNT_BACK 122 READ_EX_BACK 0 EXTENSIONS_BACK BEST_EXTENSION_BACK

5B yes LASTCHANCE yes REGEX_BACK 1 COUNTSEED FINISH2

12

1811841 SEED_exists

917 READ_LENGTH 500 INSERT_SIZE

701 POSITION 216 POSITION_BACK LAST_CHANCE_BACK CCATATTTTTCTGTTAAAGATACAATAAGATGT READ_END CCACTTCTCATAATAAAACAAGACTCAATTATA READ_START 98 MATCH_ARRAY_READ 243 MATCH_ARRAY_BACK_READ

81 READ_COUNT 81 READ_EX 0 EXTENSIONS 78 AVERAGE_COVERAGE BEST_EXTENSION

OPTION1

210 READ_COUNT_BACK 0 READ_EX_BACK 210 EXTENSIONS_BACK GTTTTATTTATATACATATCAAATGTGGCCTCTAATGAAAAATTCTATATAACAAGCAAAAACTCATTATTAATTTTATTAATT BEST_EXTENSION_BACK

yes LASTCHANCE_BACK 1 COUNTSEED FINISH2

13

1811841 SEED_exists

1001 READ_LENGTH 500 INSERT_SIZE

701 POSITION 300 POSITION_BACK USE_REGEX CCATATTTTTCTGTTAAAGATACAATAAGATGT READ_END GTTTTATTTATATACATATCAAATGTGGCCTCT READ_START 107 MATCH_ARRAY_READ 116 MATCH_ARRAY_BACK_READ yes USE_REGEX_REVERSE

95 READ_COUNT 91 READ_EX 0 EXTENSIONS 72 AVERAGE_COVERAGE BEST_EXTENSION

OPTION3

99 READ_COUNT_BACK 99 READ_EX_BACK 0 EXTENSIONS_BACK BEST_EXTENSION_BACK

2B 1 COUNTSEED FINISH2

14

1811841 SEED_exists

1001 READ_LENGTH 500 INSERT_SIZE

701 POSITION 300 POSITION_BACK SNP_ACTIVE USE_REGEX USE_REGEX_BACK2 CCATATTTTTCTGTTAAAGATACAATAAGATGT READ_END GTTTTATTTATATACATATCAAATGTGGCCTCT READ_START 107 MATCH_ARRAY_READ 133 MATCH_ARRAY_BACK_READ yes USE_REGEX_REVERSE 4 TIME1

95 READ_COUNT 91 READ_EX 0 EXTENSIONS 67 AVERAGE_COVERAGE BEST_EXTENSION

OPTION3b USE_REGEX_BACK_REVERSE

117 READ_COUNT_BACK 117 READ_EX_BACK 0 EXTENSIONS_BACK BEST_EXTENSION_BACK

5B yes REGEX_BACK 1 COUNTSEED FINISH2

15

1811841 SEED_exists

1001 READ_LENGTH 500 INSERT_SIZE

701 POSITION 300 POSITION_BACK LAST_CHANCE LAST_CHANCE_BACK USE_REGEX CCATATTTTTCTGTTAAAGATACAATAAGATGT READ_END GTTTTATTTATATACATATCAAATGTGGCCTCT READ_START 236 MATCH_ARRAY_READ 235 MATCH_ARRAY_BACK_READ yes USE_REGEX_REVERSE 6 TIME1

211 READ_COUNT 0 READ_EX 205 EXTENSIONS 76 AVERAGE_COVERAGE L BEST_EXTENSIONll 79 A 0 C 0 T 126 G 79 A 0 C 0 T 126 G 2 COUNT_SPLIT GROUP2 GTAATAATACTATTGTTTTTT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTC G GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATC GTAATAATACTATTGTTTTTTATATTAATAAACACT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCCATATTAGCAGC GTAATAATACTATTGTTTTTT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCA GTAATAATACTATTGTTTTTTATATT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGG GTAATAATACTATTGTTTTTTATATTAATAAACACTATACGATAACTCGCAACCGAGGCTTCAATTTGTAT GTAATAATACTATTGTTTTTTATATT GTAATAATACTTTTGTTTTTTATATTAATAAACACTATAGAACCTCAGATTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTTGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAAC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCCATATTAGCAGCAATCAGAATACTA GTAATAATACTATTGTTTTT GTAAT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAAT GTAATAATACTATTGTTTTTTATATTAATAAA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTATAGGAGATCCAGAAAACTATATTACAGAAAATAAATGAGTAACGCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATAGGCAATTCTACGATCTATCCCTAATAAAAT GTAATAATA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCTAGCAAATCCATTAGT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATC GTAATAAT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAAC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACAT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAG GTAATAATACTATTGTTTTTTATATTAATAAACAC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCCATATTAGCAGCAATC GTAATAATACTATTGTTT GTAATAATACTATTGTTTTTTATATTAATAAACACTAT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCACCTTCTAGGACATCCAGAAAACAATATTCCACCAAATCCATTATTAACTCCTGTTCACATCCA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTAT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGAGTAATCGCCATATTAGCAGCA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAG GTAATAATACT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTAT GTAATAATACTATTGTTTTTTATATT GTAATAATACTATTGTTCTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGC GTAATAATACTAT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAA GTAATAATACTATTGTTTTTTATATTAATAAAC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATCTTTTCT GTAATAATACTATTGTTTT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGAAACTCCTGTTCACATC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTA GTAATAATACTATTGTTTTTTATATTAATAAACAC GTAATAATACT GTAA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTC GTAATAATACTAT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCC GTAATAATACTATTTTTTTTTATATTACTAA GTAATAATACTATTGTTTTTTATATTAAT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTGTATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATG GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGG GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAG GTAATAATACTATTGTTTTTTATATTA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTATG GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAG GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGG GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCC GTAATAATACTATTGTTTTTTAT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCG GTAATAATACTAT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATACCTAATAAAATAGGAGCAGTAATCGCCATATTAGC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCG GTAATACTAT GTAATAATACTAT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCCATATTAGC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAAC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCTTTAGTAACTCCTGTTCCCATCCA GTAATAATACTATTGTTT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCG GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCGCAGCTTCTAGGAGATCCAGAAAACTATATTCCAG GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCCATATTAGCAGCAATCA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAG GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAAC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCCATATTAGC GTAATAATACTATT GTAATAATACTATTGTTTTTTATATTAATAAAAACTAT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAG GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTAT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACC GTAATAATACTATTGTTTTTTATAGATCGGGAAGAGCACACGTCTGAACTCCAGTCACACTGATATATCTCGT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCT GTAATAATACTATTGTTTTTTATATTAATAAACACTAT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCTGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCACTTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCCATATTAGCAGCAATCAGAATACT GTAATAATACTAGTGTTTATTAATATAAAAAACAATAGTATTATTGCACATCTTATTGTATCTTTAACAGAAAAATATGGGTGAAATGGAATTTTATCAGTATTACTATCTACCCC GTAATAATAATATTGTTTTTTATATTAATAAACACT GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCTATTAGTAACTCCTGTTC GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAA GTAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAAC BEST_EXTENSION2

GROUP1 ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAAT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCCATATTAGCAGCAATC ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGC ATAATAATAC ATAATAATACTATTGT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTC ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCA ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAAT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAAGCTGAGGTTCTATAGTGTTTATTAATA ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCCATATTAGCAGCAATCAGAATAAT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTC ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTGCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACG ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTATAGGAGATCTAGAAAAC ATAATAATACTATTGTTTTTTATATTAATAAACAC ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTACAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTAT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCCTTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCCATATTAG ATAATAATACTT ATAATAATACTATTGTTTTTTATATTAAGAAACACTATAGAACCTCAGCT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCA ATAATAATAC ATAATAATACTATTATTTTTTATATTAATAAACACTATAGAACCTCAACTTCTAGGGTACC ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCAAACCAGAATGATATATTCTATTTGCATACGCACT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACA ATAATAATACT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCCATA ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCC ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAAC ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCCGCTTCTAGGAGATCC ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACTTCAGCTTT ATAATAATACTATTGTTTTTTATATTAATAAATACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGTTATTTACTATTTGCATACGCCATTCTAATATC ATAATAATA ATAATAATACTATTGTTTTTTATATTAATAAACACTATAG ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGGAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCG ATAATAATACTATT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTA ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCTGAAAACTATATTCCAGC ATAATAATACTATTTTTTTTTATATTAATAAACCCTATAGAACCTCAC ATAATAATACTATTGTTTTTTATGTTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCATGTTCACATCCAATCAGCATGATATTTTCTACTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAAGAGGTGTAATCTCCATATTATGACCAATC ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAG ATAATAATACTATTGTTTTTTATATTAATAA ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAAT ATAATAATACTATTGTTTTTTATATTAATAA ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGTAGATCCAGAAAACTATATTCCAGCAAAT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCCA ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAG ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCCATATTAGCAGCAATCAGAAT ATAATAATACTATTGTTTTTTATAT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCCATAT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCC ATAATAATACTATTGTTTTTTAT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAG ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGG ATAATAATACTATTGTTTTTTATATTAATAAAC ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCAC ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCTAGCAAATC ATAATAATACTATTGTTTTTTA ATAATAATACAATTGTTTTTTATATTATTAAACACTATAGAACCTTAGCTTCTAGACGATCCAGAAAACTATATTCCAGAAAATCCATTAGTAACTCATGTTCACATCCAACCAGAAGGTTCTTTTCTATTTGCATACGTAATTCTACGATCCATCCCTATTAAAATAGGAGGTGTAAGCGAAAGATTAGC ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAG ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCC ATAATAATACTATTATTTTTTATATTAATAAACACTATAGAACCTCAACTTCTAGGGGACCCTGAAAACTTTATTCCAGCAAACCCATTAGTGACCCCTATCCATATCCAACCAGAATGATACTTTTTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGGGGTGTAATCGCCATATT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACG ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCCATATTAGC ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTG ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATT ATAATAATACTATTTTTTTTTATATTACTAAACACTATAGAACCTCAGCTTCTAGGAGATCCGGAAAACTATATTACAGCAAATCCAATCGTAACCC A ATAATAATACTATTGTTTTTTA ATA ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCTAATAAAATAGGAGGTGTAATCGCC ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTC ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGCCAATTGAACTCACCTCACCAAAAAACATTT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATTAGTAACTCCTGTTCACATCCAACCAGAATGATATTTTCTATTTGCATACGCAATTCTACGATCTATCCCT ATAATAATACTATTGTTTTTTATATTAATAAACACTATAGAACCTCAGCTTCTAGGAGATCCAGAAAACTATATTCCAGCAAATCCATT BEST_EXTENSION1

10 OVERHANG 5 COUNT_TEST1 6 COUNT_TEST2 0 COUNT_TEST3 0 COUNT_TEST4 GCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT END_SHORT_TMP

TGTAGAATAACATAGAAATTGTCTTTTTATACCCACTTTACCTTAAAATAGTCATAATGATAGATGGGGATCGCCCAATAATCTCGGACAAACCACGTTCATATTATTTACTTAATAGTGATTTCGACATTAATACTTACCATATTCCTTTACATATCGCTTTTTAGCACAATTACACCGTAAAAGTTATCTTTTAGGAGGGGTATTAGTATCTTATTATAACAAGGGATGTATACCTTATTGTCTTTCATCCAAACACTAATGACAACG REVERSE_END_SHORT_TMP

1 FIRST_YUYU 3 SECOND_YUYU 0 THIRD_YUYU 0 FOURTH_YUYU 1 PAIR1 2 PAIR2 0 PAIR3 0 PAIR4 0 CHECK_STAR 4 COUNT_ALL

MAKE BEFORE SHORTER2

15 OVERHANG 8 COUNT_TEST1 13 COUNT_TEST2 0 COUNT_TEST3 0 COUNT_TEST4 GCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT END_SHORT_TMP

TGTAGAATAACATAGAAATTGTCTTTTTATACCCACTTTACCTTAAAATAGTCATAATGATAGATGGGGATCGCCCAATAATCTCGGACAAACCACGTTCATATTATTTACTTAATAGTGATTTCGACATTAATACTTACCATATTCCTTTACATATCGCTTTTTAGCACAATTACACCGTAAAAGTTATCTTTTAGGAGGGGTATTAGTATCTTATTATAACAAGGGATGTATACCTTATTGTCTTTCATCCAAACACTAATGACAACG REVERSE_END_SHORT_TMP

4 FIRST_YUYU 10 SECOND_YUYU 0 THIRD_YUYU 0 FOURTH_YUYU 1 PAIR1 6 PAIR2 0 PAIR3 0 PAIR4 0 CHECK_STAR 14 COUNT_ALL

MAKE BEFORE SHORTER2

20 OVERHANG 9 COUNT_TEST1 17 COUNT_TEST2 0 COUNT_TEST3 0 COUNT_TEST4 GCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT END_SHORT_TMP

TGTAGAATAACATAGAAATTGTCTTTTTATACCCACTTTACCTTAAAATAGTCATAATGATAGATGGGGATCGCCCAATAATCTCGGACAAACCACGTTCATATTATTTACTTAATAGTGATTTCGACATTAATACTTACCATATTCCTTTACATATCGCTTTTTAGCACAATTACACCGTAAAAGTTATCTTTTAGGAGGGGTATTAGTATCTTATTATAACAAGGGATGTATACCTTATTGTCTTTCATCCAAACACTAATGACAACG REVERSE_END_SHORT_TMP

5 FIRST_YUYU 13 SECOND_YUYU 0 THIRD_YUYU 0 FOURTH_YUYU 1 PAIR1 7 PAIR2 0 PAIR3 0 PAIR4 0 CHECK_STAR 18 COUNT_ALL

MAKE BEFORE SHORTER2

25 OVERHANG 13 COUNT_TEST1 20 COUNT_TEST2 0 COUNT_TEST3 0 COUNT_TEST4 GCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT END_SHORT_TMP

TGTAGAATAACATAGAAATTGTCTTTTTATACCCACTTTACCTTAAAATAGTCATAATGATAGATGGGGATCGCCCAATAATCTCGGACAAACCACGTTCATATTATTTACTTAATAGTGATTTCGACATTAATACTTACCATATTCCTTTACATATCGCTTTTTAGCACAATTACACCGTAAAAGTTATCTTTTAGGAGGGGTATTAGTATCTTATTATAACAAGGGATGTATACCTTATTGTCTTTCATCCAAACACTAATGACAACG REVERSE_END_SHORT_TMP

7 FIRST_YUYU 17 SECOND_YUYU 0 THIRD_YUYU 0 FOURTH_YUYU 2 PAIR1 9 PAIR2 0 PAIR3 0 PAIR4 0 CHECK_STAR 24 COUNT_ALL

MAKE BEFORE SHORTER2

30 OVERHANG 13 COUNT_TEST1 25 COUNT_TEST2 0 COUNT_TEST3 0 COUNT_TEST4 GCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT END_SHORT_TMP

TGTAGAATAACATAGAAATTGTCTTTTTATACCCACTTTACCTTAAAATAGTCATAATGATAGATGGGGATCGCCCAATAATCTCGGACAAACCACGTTCATATTATTTACTTAATAGTGATTTCGACATTAATACTTACCATATTCCTTTACATATCGCTTTTTAGCACAATTACACCGTAAAAGTTATCTTTTAGGAGGGGTATTAGTATCTTATTATAACAAGGGATGTATACCTTATTGTCTTTCATCCAAACACTAATGACAACG REVERSE_END_SHORT_TMP

7 FIRST_YUYU 22 SECOND_YUYU 0 THIRD_YUYU 0 FOURTH_YUYU 2 PAIR1 10 PAIR2 0 PAIR3 0 PAIR4 0 CHECK_STAR 29 COUNT_ALL

MAKE BEFORE SHORTER2

35 OVERHANG 17 COUNT_TEST1 30 COUNT_TEST2 0 COUNT_TEST3 0 COUNT_TEST4 GCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT END_SHORT_TMP

TGTAGAATAACATAGAAATTGTCTTTTTATACCCACTTTACCTTAAAATAGTCATAATGATAGATGGGGATCGCCCAATAATCTCGGACAAACCACGTTCATATTATTTACTTAATAGTGATTTCGACATTAATACTTACCATATTCCTTTACATATCGCTTTTTAGCACAATTACACCGTAAAAGTTATCTTTTAGGAGGGGTATTAGTATCTTATTATAACAAGGGATGTATACCTTATTGTCTTTCATCCAAACACTAATGACAACG REVERSE_END_SHORT_TMP

8 FIRST_YUYU 24 SECOND_YUYU 0 THIRD_YUYU 0 FOURTH_YUYU 2 PAIR1 11 PAIR2 0 PAIR3 0 PAIR4 0 CHECK_STAR 32 COUNT_ALL

MAKE BEFORE SHORTER2

40 OVERHANG 18 COUNT_TEST1 35 COUNT_TEST2 0 COUNT_TEST3 0 COUNT_TEST4 GCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT END_SHORT_TMP

TGTAGAATAACATAGAAATTGTCTTTTTATACCCACTTTACCTTAAAATAGTCATAATGATAGATGGGGATCGCCCAATAATCTCGGACAAACCACGTTCATATTATTTACTTAATAGTGATTTCGACATTAATACTTACCATATTCCTTTACATATCGCTTTTTAGCACAATTACACCGTAAAAGTTATCTTTTAGGAGGGGTATTAGTATCTTATTATAACAAGGGATGTATACCTTATTGTCTTTCATCCAAACACTAATGACAACG REVERSE_END_SHORT_TMP

10 FIRST_YUYU 27 SECOND_YUYU 0 THIRD_YUYU 0 FOURTH_YUYU 3 PAIR1 11 PAIR2 0 PAIR3 0 PAIR4 0 CHECK_STAR 37 COUNT_ALL

MAKE BEFORE SHORTER2

45 OVERHANG 18 COUNT_TEST1 35 COUNT_TEST2 0 COUNT_TEST3 0 COUNT_TEST4 GCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT END_SHORT_TMP

TGTAGAATAACATAGAAATTGTCTTTTTATACCCACTTTACCTTAAAATAGTCATAATGATAGATGGGGATCGCCCAATAATCTCGGACAAACCACGTTCATATTATTTACTTAATAGTGATTTCGACATTAATACTTACCATATTCCTTTACATATCGCTTTTTAGCACAATTACACCGTAAAAGTTATCTTTTAGGAGGGGTATTAGTATCTTATTATAACAAGGGATGTATACCTTATTGTCTTTCATCCAAACACTAATGACAACG REVERSE_END_SHORT_TMP

11 FIRST_YUYU 27 SECOND_YUYU 0 THIRD_YUYU 0 FOURTH_YUYU 4 PAIR1 11 PAIR2 0 PAIR3 0 PAIR4 0 CHECK_STAR 38 COUNT_ALL GTTTTATTTATATACATATCAAATGTGGCCTCTAATGAAAAATTCTATATAACAAGCAAAAACTCATTATTAATTTTATTAATTCCACTTCTCATAATAAAACAAGACTCAATTATATACAATTTACATAATTATAAAACTAATTCCACAATAAAATATGATATTTCCACCTCCTTAAGAAAGCTGTTTTCATCAGAAACAGTACCTTTAATTATAATAATAGTTTTATATTTATTCTTCACTATAATTGTTATTTCATCAATTGTAAATATTCATGAAGGGCCAATACGCTCGAAAAGATAATGAATATATCAACACGTAAAACTAACCCTTTAATTAAAACATTAAATAATTTACTTATTGATCTCCCATGTCCTACAAGAATCTCCAATTGATGAAATTTTGGATCCTTACTAAGAATATGTTTGTTAATCCAATTATTAACAGGAATCTTTTTAGCCATACATTATACAGCTAACATTGAATTAGCCTTCAACAGTGTAATTCACATTATACGTAATGTAAATAATGGTTGAATAATACGAAGTATACATGCTAACGGCGCCTCATTCTTTTTTATTTGTATATATATACATGTAGGACGAGGAATTTACTATAATTCTTATCAACTAACTAACACTTGAATAGTAGGAGTAATAATGTTATTACTAACAATAGCAACAGCCTTCCTAGGTTATGTTTTACCATGAGGACAAATGTCCCTGTGGGGGGCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT READ_SHORT 0 TESTh

REFGTTTTATTTATATACATATCAAATGTGGCCTCTAATGAAAAATTCTATATAACAAGCAAAAACTCATTATTAATTTTATTAATTCCACTTCTCATAATAAAACAAGACTCAATTATATACAATTTACATAATTATAAAACTAATTCCACAATAAAATATGATATTTCCACCTCCTTAAGAAAGCTGTTTTCATCAGAAACAGTACCTTTAATTATAATAATAGTTTTATATTTATTCTTCACTATAATTGTTATTTCATCAATTGTAAATATTCATGAAGGGCCAATACGCTCGAAAAGATAATGAATATATCAACACGTAAAACTAACCCTTTAATTAAAACATTAAATAATTTACTTATTGATCTCCCATGTCCTACAAGAATCTCCAATTGATGAAATTTTGGATCCTTACTAAGAATATGTTTGTTAATCCAATTATTAACAGGAATCTTTTTAGCCATACATTATACAGCTAACATTGAATTAGCCTTCAACAGTGTAATTCACATTATACGTAATGTAAATAATGGTTGAATAATACGAAGTATACATGCTAACGGCGCCTCATTCTTTTTTATTTGTATATATATACATGTAGGACGAGGAATTTACTATAATTCTTATCAACTAACTAACACTTGAATAGTAGGAGTAATAATGTTATTACTAACAATAGCAACAGCCTTCCTAGGTTATGTTTTACCATGAGGACAAATGTCCCTGTGGGGGGCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT NEG1__ NEG20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
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0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

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0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
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0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
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0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
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0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 COUNT1B 0 COUNT2B 0 COUNT3B 0 COUNT4B 4 COUNT1B_NO 11 COUNT2B_NO 0 COUNT3B_NO 0 COUNT4B_NO

MAKE BEFORE SHORTER3

65 OVERHANG 26 COUNT_TEST1 47 COUNT_TEST2 0 COUNT_TEST3 0 COUNT_TEST4 GCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT END_SHORT_TMP

TGTAGAATAACATAGAAATTGTCTTTTTATACCCACTTTACCTTAAAATAGTCATAATGATAGATGGGGATCGCCCAATAATCTCGGACAAACCACGTTCATATTATTTACTTAATAGTGATTTCGACATTAATACTTACCATATTCCTTTACATATCGCTTTTTAGCACAATTACACCGTAAAAGTTATCTTTTAGGAGGGGTATTAGTATCTTATTATAACAAGGGATGTATACCTTATTGTCTTTCATCCAAACACTAATGACAACG REVERSE_END_SHORT_TMP

19 FIRST_YUYU 39 SECOND_YUYU 0 THIRD_YUYU 0 FOURTH_YUYU 8 PAIR1 17 PAIR2 0 PAIR3 0 PAIR4 0 CHECK_STAR 58 COUNT_ALL GTTTTATTTATATACATATCAAATGTGGCCTCTAATGAAAAATTCTATATAACAAGCAAAAACTCATTATTAATTTTATTAATTCCACTTCTCATAATAAAACAAGACTCAATTATATACAATTTACATAATTATAAAACTAATTCCACAATAAAATATGATATTTCCACCTCCTTAAGAAAGCTGTTTTCATCAGAAACAGTACCTTTAATTATAATAATAGTTTTATATTTATTCTTCACTATAATTGTTATTTCATCAATTGTAAATATTCATGAAGGGCCAATACGCTCGAAAAGATAATGAATATATCAACACGTAAAACTAACCCTTTAATTAAAACATTAAATAATTTACTTATTGATCTCCCATGTCCTACAAGAATCTCCAATTGATGAAATTTTGGATCCTTACTAAGAATATGTTTGTTAATCCAATTATTAACAGGAATCTTTTTAGCCATACATTATACAGCTAACATTGAATTAGCCTTCAACAGTGTAATTCACATTATACGTAATGTAAATAATGGTTGAATAATACGAAGTATACATGCTAACGGCGCCTCATTCTTTTTTATTTGTATATATATACATGTAGGACGAGGAATTTACTATAATTCTTATCAACTAACTAACACTTGAATAGTAGGAGTAATAATGTTATTACTAACAATAGCAACAGCCTTCCTAGGTTATGTTTTACCATGAGGACAAATGTCCCTGTGGGGGGCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT READ_SHORT 0 TESTh

REFGTTTTATTTATATACATATCAAATGTGGCCTCTAATGAAAAATTCTATATAACAAGCAAAAACTCATTATTAATTTTATTAATTCCACTTCTCATAATAAAACAAGACTCAATTATATACAATTTACATAATTATAAAACTAATTCCACAATAAAATATGATATTTCCACCTCCTTAAGAAAGCTGTTTTCATCAGAAACAGTACCTTTAATTATAATAATAGTTTTATATTTATTCTTCACTATAATTGTTATTTCATCAATTGTAAATATTCATGAAGGGCCAATACGCTCGAAAAGATAATGAATATATCAACACGTAAAACTAACCCTTTAATTAAAACATTAAATAATTTACTTATTGATCTCCCATGTCCTACAAGAATCTCCAATTGATGAAATTTTGGATCCTTACTAAGAATATGTTTGTTAATCCAATTATTAACAGGAATCTTTTTAGCCATACATTATACAGCTAACATTGAATTAGCCTTCAACAGTGTAATTCACATTATACGTAATGTAAATAATGGTTGAATAATACGAAGTATACATGCTAACGGCGCCTCATTCTTTTTTATTTGTATATATATACATGTAGGACGAGGAATTTACTATAATTCTTATCAACTAACTAACACTTGAATAGTAGGAGTAATAATGTTATTACTAACAATAGCAACAGCCTTCCTAGGTTATGTTTTACCATGAGGACAAATGTCCCTGTGGGGGGCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT NEG1__ NEG20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
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0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
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0 COUNT1B 0 COUNT2B 0 COUNT3B 0 COUNT4B 8 COUNT1B_NO 17 COUNT2B_NO 0 COUNT3B_NO 0 COUNT4B_NO

MAKE BEFORE SHORTER3

85 OVERHANG 37 COUNT_TEST1 60 COUNT_TEST2 0 COUNT_TEST3 0 COUNT_TEST4 GCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT END_SHORT_TMP

TGTAGAATAACATAGAAATTGTCTTTTTATACCCACTTTACCTTAAAATAGTCATAATGATAGATGGGGATCGCCCAATAATCTCGGACAAACCACGTTCATATTATTTACTTAATAGTGATTTCGACATTAATACTTACCATATTCCTTTACATATCGCTTTTTAGCACAATTACACCGTAAAAGTTATCTTTTAGGAGGGGTATTAGTATCTTATTATAACAAGGGATGTATACCTTATTGTCTTTCATCCAAACACTAATGACAACG REVERSE_END_SHORT_TMP

27 FIRST_YUYU 46 SECOND_YUYU 0 THIRD_YUYU 0 FOURTH_YUYU 12 PAIR1 21 PAIR2 0 PAIR3 0 PAIR4 0 CHECK_STAR 73 COUNT_ALL GTTTTATTTATATACATATCAAATGTGGCCTCTAATGAAAAATTCTATATAACAAGCAAAAACTCATTATTAATTTTATTAATTCCACTTCTCATAATAAAACAAGACTCAATTATATACAATTTACATAATTATAAAACTAATTCCACAATAAAATATGATATTTCCACCTCCTTAAGAAAGCTGTTTTCATCAGAAACAGTACCTTTAATTATAATAATAGTTTTATATTTATTCTTCACTATAATTGTTATTTCATCAATTGTAAATATTCATGAAGGGCCAATACGCTCGAAAAGATAATGAATATATCAACACGTAAAACTAACCCTTTAATTAAAACATTAAATAATTTACTTATTGATCTCCCATGTCCTACAAGAATCTCCAATTGATGAAATTTTGGATCCTTACTAAGAATATGTTTGTTAATCCAATTATTAACAGGAATCTTTTTAGCCATACATTATACAGCTAACATTGAATTAGCCTTCAACAGTGTAATTCACATTATACGTAATGTAAATAATGGTTGAATAATACGAAGTATACATGCTAACGGCGCCTCATTCTTTTTTATTTGTATATATATACATGTAGGACGAGGAATTTACTATAATTCTTATCAACTAACTAACACTTGAATAGTAGGAGTAATAATGTTATTACTAACAATAGCAACAGCCTTCCTAGGTTATGTTTTACCATGAGGACAAATGTCCCTGTGGGGGGCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT READ_SHORT 0 TESTh

REF__GTTTTATTTATATACATATCAAATGTGGCCTCTAATGAAAAATTCTATATAACAAGCAAAAACTCATTATTAATTTTATTAATTCCACTTCTCATAATAAAACAAGACTCAATTATATACAATTTACATAATTATAAAACTAATTCCACAATAAAATATGATATTTCCACCTCCTTAAGAAAGCTGTTTTCATCAGAAACAGTACCTTTAATTATAATAATAGTTTTATATTTATTCTTCACTATAATTGTTATTTCATCAATTGTAAATATTCATGAAGGGCCAATACGCTCGAAAAGATAATGAATATATCAACACGTAAAACTAACCCTTTAATTAAAACATTAAATAATTTACTTATTGATCTCCCATGTCCTACAAGAATCTCCAATTGATGAAATTTTGGATCCTTACTAAGAATATGTTTGTTAATCCAATTATTAACAGGAATCTTTTTAGCCATACATTATACAGCTAACATTGAATTAGCCTTCAACAGTGTAATTCACATTATACGTAATGTAAATAATGGTTGAATAATACGAAGTATACATGCTAACGGCGCCTCATTCTTTTTTATTTGTATATATATACATGTAGGACGAGGAATTTACTATAATTCTTATCAACTAACTAACACTTGAATAGTAGGAGTAATAATGTTATTACTAACAATAGCAACAGCCTTCCTAGGTTATGTTTTACCATGAGGACAAATGTCCCTGTGGGGGGCAACAGTAATCACAAACCTACTTTCTGTTATTCCATATGTAGGGAACAATATTATTCTATGATTATGGGGAGGATTTTCTATTGAAAATGCCACATTAACACGATTTTTCGCTATACATTTCCTTATACCATTCATAATTACAGCTTTAGTGATAATTCATTTATTATACTTGCACCAAACAGGCTCTAATAACCCGCTAGGGGTAGATAGTAATACTGATAAAATTCCATTTCACCCATATTTTTCTGTTAAAGATACAATAAGATGT NEG1___

ndierckx commented 3 years ago

hi,

Can't you send them as files, you can just drag the text files in the comment field

brenna-levine commented 3 years ago

Darn! I thought I had attached them. I'll try it again here. Thanks for your patience. log_H-C-M1_S10.txt

brenna-levine commented 3 years ago

log_B-U-M1_S21.txt log_B-D-F1_S15.txt

ndierckx commented 3 years ago

Ok, there is something weird with your data of H-C-M1_S10, it doesn't seem to find any matches for the paired reads? Are they paired reads and insert size is around 500?

brenna-levine commented 3 years ago

They are paired. The insert size if between 500-1000 bp. I have inherited these data from some previous researchers in my lab. I think they may have been trimmed, but I can't confirm.

Brenna

brenna-levine commented 3 years ago

These were whole shotgun reads.

ndierckx commented 3 years ago

Can try to run it with insert size of 1000 maybe, because it doesn't use paired read information which is crucial for a good assembly. Although if you put 500 as insert size it should have found some paired reads because margin is relativly big. Are they mate pairs in stead of paired reads maybe? I never saw this problem so it's weird

log_B-U-M1_S21.txt, this file is just a normal log file, not the extended log file

log_B-D-F1_S15.txt This dataset works better, but as expected it has a large repetitive control region, you use this often in insects. So it is probably not linear, just a complex control region that can not be assembled with short reads. similar as in the paper of NOVOPlasty with our beetle genome. I is probably circular because both sides end up in the repetitive region.

And the fact it doesn't match other assemblers contigs or NCBI reference genome is normal, almost all mitogenomes with repetitive control regions are incomplete on NCBI. NOVOPlasty assembles more, but because the complexity of the control region it could be that it makes the repetitive control region is too long (but could be too short too). You can trust more than 12000bp, that's for sure. I would even trust the first 17500 bp.. This repetitive control regions can also differ a lot between individuals so hard to compare.......

As for heteroplasmy analysis, make sure you don't include any repetitive region. But there are lot of duplicated regions so need to cut a lot from the 18000bp

Greets,

Nicolas

brenna-levine commented 3 years ago

Hi Nicolas,

Thanks for looking through my files. Apologies that I sent a normal log file rather than extended for one of the samples.

A couple quick questions for you: (1) I don't thing they are mate pair. If they are, how does that change interpretation? (2) The mitogenome should only be 15,000-16,000 bp long. How can we then interpret a 17,500 bp long contig? (3) Do you have any recommendations for a different program that you think we should be using to get at this problem?

Thanks again for all of your insight. If we can't use NOVOplasty for this particular project, we will be sure to use it in the future.

Brenna

brenna-levine commented 3 years ago

Hi again, Nicolas;

I wanted to attach one more extended log file for you.

Can you also provide some information on how you can determine that both sides are ending up in the repetitive region and what our coverage is (for instance, line numbers in the extended log?).

Brenna log_extended_bed_bug.txt

ndierckx commented 3 years ago

(1) I don't have an option for mate pairs so it would use it as single reads, like it did with this assembly. Other possible causes are different orientation of the reads than standard paired end, or a problem of the read ids...

(2) How you know it should be only that long?

(3) I haven't use it it before but getorganelle is a new assembly tool. But I don't think other tools will give you a complete assembly. These kind of repetitive regions can not be assembled with short reads. Other tools will maybe give you a nice short contig, but that is incorrect. I found so many incorrect assemblies where repetitive region are collapsed completely. If you really want the complete genome, you need long reads

Greets,

Nicolas

ndierckx commented 3 years ago

You can try also spades, and check what the largest contig looks like

ndierckx commented 3 years ago

Of the third file coverage is around 800 I just checked the fasta file manually, you can see the both sides of the assembly are repetitive, easy by seach and find in text editor

brenna-levine commented 3 years ago

Thanks Nicolas, There are two reference mitogenomes for this species, both of which are between 15-16,000 bp. However, the methods by which these were constructed are very vague, and we're not sure how much confidence to have in them.

Thanks for this information. Where in the extended log file does it say the depth of coverage?

Brenna

ndierckx commented 3 years ago

Is the average coverage not given in the normal log file?

And I would not trust any assembly when there is a repetitive control region. You can check the paper of NOVOPlasty. We resolved the mitogenomes of gonioctena intermedia only by using long reads. It's often impossible for any assembler and as it is repetitive, it can be circularized at many positions, so very complex. Plus we observed significant difference between individuals.

For heteroplasmy analysis, you can look at the heteroplasmy paper of NOVOPlasty, as we had a similar situation

brenna-levine commented 3 years ago

Hi Nicolas,

Ah - I see where coverage is. Thanks. I appreciate all of your help.

Brenna

brenna-levine commented 3 years ago

One final question for you and then I'll close the issue.

What is your stance on assembling mitogenomes from RADseq data using your program? Any tricks or pitfalls to be aware of?

Brenna

ndierckx commented 3 years ago

No idea how RAD seq data will look like. But if there are gaps in coverage across the genome and you want to assemble different contigs, I would go for a graph based tool and not NOVOPlasty (it needs reads across the whole genome or multiple seeds, but that is more effort)

brenna-levine commented 3 years ago

Thanks, Nicolas.

Brenna