V4 region use Closed_reference only 1 OTU. Error?

Sophie_Liu

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Jun 20, 2017, 10:15:13 PM6/20/17

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Hello,

i have a set of V4 region data, length distribition were as follow:

22 169

1 208

6 209

3 212

1 220

42 221

5 223

1 230

7 231

2 248

2 249

315 250

4210 251

234204 252

711903 253

28204 254

457 255

22 256

1 257

3 258

25 259

1 26

837 260

154 261

1 262

6 263

34 264

3 265

1 267

80 268

12 269

1 272

1 275

1 276

1 277

280 278

7 279

4 280

16 281

1 283

2 284

3 285

2 286

11 287

11 288

12 289

2 290

3 291

1 297

12 299

1 303

2 56

I want to make otu table use Closed_reference method, my scripts were:

pick_closed_reference_otus.py -i seqs_no_chimera.fna -r $PATH/Silva_123/rep_set/rep_set_16S_only/97/97_otus_16S.fasta -t $PATH/18S_Silva/Silva_123/taxonomy/16S_only/97/taxonomy_2016.11.15.txt -o otu_w_tax

But the result shows only one OTU.

======== seqs_no_chimera_clusters.uc ================================

N * 252 * * * * * QiimeExactMatch.s10_9692 *

N * 253 * * * * * QiimeExactMatch.s10_5074 *

N * 253 * * * * * QiimeExactMatch.s10_1997 *

N * 253 * * * * * QiimeExactMatch.s10_6 *

N * 253 * * * * * QiimeExactMatch.s10_553 *

N * 253 * * * * * QiimeExactMatch.s10_2 *

N * 252 * * * * * QiimeExactMatch.s10_2747 *

N * 252 * * * * * QiimeExactMatch.s10_737 *

N * 253 * * * * * QiimeExactMatch.s10_1898 *

N * 253 * * * * * QiimeExactMatch.s10_3982 *

N * 252 * * * * * QiimeExactMatch.s10_4704 *

N * 253 * * * * * QiimeExactMatch.s10_7 *

N * 253 * * * * * QiimeExactMatch.s16_17364 *

N * 253 * * * * * QiimeExactMatch.s10_9203 *

N * 252 * * * * * QiimeExactMatch.s10_3023 *

N * 253 * * * * * QiimeExactMatch.s11_9520 *

N * 253 * * * * * QiimeExactMatch.s10_18614 *

L 207520 1462 * * * * * JRRC01144346.928.2389 *

=================== number of OTU ========================

less uclust_ref_picked_otus/seqs_no_chimera_otus.txt | wc -l [ 9:50AM]

1

===== reads in OTUs =================================================

Counts/sample detail:

sP1: 2.0

s11: 2.0

s16: 2.0

s18: 3.0

s49: 3.0

s46: 4.0

s10: 4.0

s12: 5.0

s17: 12.0

s6: 27.0

s8: 31.0

s5: 35.0

s38: 46.0

s7: 88.0

sP2: 122.0

s19: 141.0

s3: 200.0

s47: 336.0

s9: 965.0

anyone knows the problem??

Thanks!!!

sophie

justink

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Jun 21, 2017, 5:34:31 PM6/21/17

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My guess is it's the wrong DNA strand:

"

**Note:** If most or all of your sequences are failing to hit the reference,

your sequences may be in the reverse orientation with respect to your reference

database. To address this, you should add the following line to your parameters

file (creating one, if necessary) and pass this file as -p:

pick_otus:enable_rev_strand_match True

Be aware that this doubles the amount of memory used.

"

Otherwise, maybe try:

1. blasting 2 or so of your seqs against ncbi online—make sure they hit 16S

2. download some example seqs from the qiime tutorial, and make sure they hit the silva database you're using:

pick_closed_reference_otus.py -i tutorial_seqs.fna -r $PATH/Silva_123/rep_set/rep_set_16S_only/97/97_otus_16S.fasta -t $PATH/18S_Silva/Silva_123/taxonomy/16S_only/97/taxonomy_2016.11.15.txt -o otu_w_tax

Sophie_Liu

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Jun 21, 2017, 8:53:49 PM6/21/17

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Hi justink,

thank you!

you are right. My sequences were in the reverse orientation. When i allowed reverse blast with -z parameter, the result were perfect.