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workflow_20190411.rtf
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workflow_20190411.rtf
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{\rtf1\ansi\ansicpg1252\cocoartf1504\cocoasubrtf830
{\fonttbl\f0\fnil\fcharset0 HelveticaNeue;\f1\fswiss\fcharset0 ArialMT;\f2\fswiss\fcharset0 Helvetica;
}
{\colortbl;\red255\green255\blue255;\red255\green255\blue255;\red52\green52\blue52;\red26\green26\blue26;
}
{\*\expandedcolortbl;;\cssrgb\c100000\c100000\c100000;\cssrgb\c26667\c26667\c26667;\cssrgb\c13333\c13333\c13333;
}
\margl1440\margr1440\vieww14420\viewh15120\viewkind0
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f0\b\fs24 \cf0 \
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 Data uploaded to public repositories: \
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i0 \cf0 \
NCBI Sequence Read Archive: SUB5380350\
\b0 ## Illumina MiSeq 300bp paired-end \
## split across three runs:
\b \
\
BioProject : PRJNA530061 (submission ID: SUB5380324) (same for SRA and GenBank)\
\pard\pardeftab720\partightenfactor0
\f1\b0\fs28\fsmilli14400 \cf0 \cb2 \expnd0\expndtw0\kerning0
BioProject: 530061
\f0\b\fs24 \cb1 \kerning1\expnd0\expndtw0 \
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\cf0 \
BioSample: metagenome, environmental template \'97 metagenome for each gene region\
\b0 Concatenated gene regions across runs:\
> cat ../DoubleBarcodeRuns_2019_141219/ITS_R1.fastq.gz ../DoubleBarcodeRuns_2019_150807/ITS_R1.fastq.gz ../DoubleBarcodeRuns_2019_160222/ITS_R1.fastq.gz > Sawtooth_ITS_R1.fastq.gz\
\
> cat ../DoubleBarcodeRuns_2019_141219/trnL_cf_R1.fastq.gz ../DoubleBarcodeRuns_2019_150807/trn_cf_R1.fastq.gz > Sawtooth_trn_cf_R1.fastq.gz\
\
NOTE: changed Sawtooth_trn_cf* => Sawtooth_trn_cd*\
\
\
\b Dryad:
\b0 \
TotalDataGeneRegionAlignments.zip :
\f2\fs30 \cf3 \cb2 \expnd0\expndtw0\kerning0
(06_geneTreesRemoved)\
Alignments of total sequence data pre-cleaning. Zip file includes six gene regions (atpB, ITS, matK, ndhF, rbcL, and trnT-F).\
\f0\b\fs24 \cf0 \cb1 \kerning1\expnd0\expndtw0 \
\b0 sawtooth.totalData.clean.concat :\
Alignment of cleaned total combined dataset (
\f2 MiSeq and GenBank sequences) used for community phylogeny inference.
\f0 \
\b \
\b0 speciespersummit_collect :
\b \
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f2\b0 \cf0 Alpine community matrix (rows = plant species collected, columns = summit) and indication if collected from high-alpine meadow (\'93Meadow\'94 = 1).
\f0\b \
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\b0 \cf0 sawtooth.totalData : \
Community phylogeny used for estimation of phylogenetic structure within and between nine high-alpine summits.\
\b \
\
GenBank: \
\b0 Submission IDs:\
atpB \'97
\f1\fs26 \cf4 \cb2 \expnd0\expndtw0\kerning0
2213522: MK800171 - MK800415
\f0\fs24 \cf0 \cb1 \kerning1\expnd0\expndtw0 \
ITS \'97
\f1\fs26 \cf4 \cb2 \expnd0\expndtw0\kerning0
2215112: MK802340 - MK802514
\f0\fs24 \cf0 \cb1 \kerning1\expnd0\expndtw0 \
matK \'97
\f1\fs26 \cf4 \cb2 \expnd0\expndtw0\kerning0
2213828
\fs27\fsmilli13600 \cf0 :
\fs26 \cf4 MK800416 - MK800614
\fs27\fsmilli13600 \cf0
\f0\fs24 \cb1 \kerning1\expnd0\expndtw0 \
ndhF \'97
\f1\fs26 \cf4 \cb2 \expnd0\expndtw0\kerning0
2213954
\fs27\fsmilli13600 \cf0 :
\fs26 \cf4 MK800615 - MK800980
\f0\fs24 \cf0 \cb1 \kerning1\expnd0\expndtw0 \
rbcL \'97
\f1\fs27\fsmilli13600 \cb2 \expnd0\expndtw0\kerning0
2209813:
\fs26 \cf4 MK749304 - MK749388
\f0\fs24 \cf0 \cb1 \kerning1\expnd0\expndtw0 \
trnTLF \'97
\f1\fs26 \cf4 \cb2 \expnd0\expndtw0\kerning0
2213962: MK800981 - MK801096
\f0\b\fs24 \cf0 \cb1 \kerning1\expnd0\expndtw0 \
\
\b0 \
\
################################################################################################################\
\
\b ** These are extra notes on the workflow for Marx et al. AJB 2019. Most are specific commands used, but there are also notes about decisions to remove taxa from gene trees and updates to taxonomy. **\
\
\b0 ################################################################################################################\
\b \
01_MiSeq/\
\b0 \
\b 1_preprocess: Demultiplex each of the three plates/runs (dbcAmplicons preprocess)\
Three runs:
\b0 (141219, 150807, 160222)
\b \
\b0 == id R1 and R2 by project\
\
$ dbcAmplicons preprocess -B metadata/dbcBarcodeLookupTable.txt -P metadata/PrimerTable.txt -S metadata/SawtoothMiSeq_samplesheet_141219_2019.txt -1 DoubleBarcodeRun_141219_NoIndex_L001_R1_001.fastq.gz -2 DoubleBarcodeRun_141219_NoIndex_L001_R2_001.fastq.gz -3 DoubleBarcodeRun_141219_NoIndex_L001_R3_001.fastq.gz -4 DoubleBarcodeRun_141219_NoIndex_L001_R4_001.fastq.gz -O DoubleBarcodeRuns_2019_141219\
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 Output:
\i0 \
\i 22000562 reads processed in 22.74 minutes, 577986 (2.6%) identified 20703948 reads (94.1% of total run) successfully identified barcode \
14934270 reads (67.9% of total run) successfully identified barcode and primer \
577986 reads (2.6% of total run) successfully assigned to sample \
21422576 reads (97.4% of total run) unidentified 215636 reads (1.0% of total run) found for project trnL_cf \
362350 reads (1.6% of total run) found for project ITS
\i0 \
\
\
$ dbcAmplicons preprocess -B metadata/dbcBarcodeLookupTable.txt -P metadata/PrimerTable.txt -S metadata/SawtoothMiSeq_samplesheet_150807_2019.txt -1 DoubleBarcodeRun_150807_NoIndex_L001_R1_001.fastq.gz -2 DoubleBarcodeRun_150807_NoIndex_L001_R2_001.fastq.gz -3 DoubleBarcodeRun_150807_NoIndex_L001_R3_001.fastq.gz -4 DoubleBarcodeRun_150807_NoIndex_L001_R4_001.fastq.gz -O DoubleBarcodeRuns_2019_150807\
\
\i Output:
\i0 \
\i 15089062 reads processed in 20.73 minutes, 355772 (2.4%) identified 12532691 reads (83.1% of total run) successfully identified barcode \
2810332 reads (18.6% of total run) successfully identified barcode and primer \
355772 reads (2.4% of total run) successfully assigned to sample \
14733290 reads (97.6% of total run) unidentified 117131 reads (0.8% of total run) found for project trn_ab \
47335 reads (0.3% of total run) found for project ITS \
105003 reads (0.7% of total run) found for project trn_cf \
86303 reads (0.6% of total run) found for project rbcL \
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i0 \cf0 \
\
$ dbcAmplicons preprocess -B metadata/dbcBarcodeLookupTable.txt -P metadata/PrimerTable.txt -S metadata/SawtoothMiSeq_samplesheet_160222_2019.txt -1 DoubleBarcodeRun_160222_NoIndex_L001_R1_001.fastq.gz -2 DoubleBarcodeRun_160222_NoIndex_L001_R2_001.fastq.gz -3 DoubleBarcodeRun_160222_NoIndex_L001_R3_001.fastq.gz -4 DoubleBarcodeRun_160222_NoIndex_L001_R4_001.fastq.gz -O DoubleBarcodeRuns_2019_160222\
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 Output:\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i0 \cf0 19847822 reads processed in 29.2 minutes, 622485 (3.1%) identified\
\
19026840 reads (95.9% of total run) successfully identified barcode\
626054 reads (3.2% of total run) successfully identified barcode and primer\
622485 reads (3.1% of total run) successfully assigned to sample\
19225337 reads (96.9% of total run) unidentified\
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 109843 reads (0.6% of total run) found for project ndhF_1318_3\
117064 reads (0.6% of total run) found for project matK_3F_3R\
19024 reads (0.1% of total run) found for project atpB_S1494R_S611\
106621 reads (0.5% of total run) found for project matK_1F_4R\
51256 reads (0.3% of total run) found for project atpB_S766R_S3\
113408 reads (0.6% of total run) found for project ndhF_803_1318\
93099 reads (0.5% of total run) found for project ndhF_5_803\
12170 reads (0.1% of total run) found for project ITS
\i0 \
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\b \cf0 2_screen: screen to reference seqs (dbcAmplicaons screen)\
\b0 not just fungal seqs (ITS); there is a lot of unspecific amplification\
\'97mapped\
\'97nomap\
\
\b DoubleBarcodeRuns_2019_141219/
\b0 \
$ dbcAmplicons screen -1 trnL_cf_R1.fastq.gz -2 trnL_cf_R2.fastq.gz -O screen/trn_cd_141219 -R ../refSeqs/trnL-trnF.keep\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 Output:
\i0 \
\i Processed: 100000, PE in ref: 7135, SE in ref: 0\
Processed: 200000, PE in ref: 12321, SE in ref: 0\
Processed: 300000, PE in ref: 22739, SE in ref: 0\
Processed: 400000, PE in ref: 31783, SE in ref: 0\
215636 reads; of these:\
215636 (100.00%) were paired; of these:\
190465 (88.33%) aligned concordantly 0 times\
13004 (6.03%) aligned concordantly exactly 1 time\
12167 (5.64%) aligned concordantly >1 times\
----\
190465 pairs aligned concordantly 0 times; of these:\
179 (0.09%) aligned discordantly 1 time\
----\
190286 pairs aligned 0 times concordantly or discordantly; of these:\
380572 mates make up the pairs; of these:\
370837 (97.44%) aligned 0 times\
6136 (1.61%) aligned exactly 1 time\
3599 (0.95%) aligned >1 times\
14.01% overall alignment rate\
Records processed: 431272, PE in ref: 34253, SE in ref: 0
\i0 \
\
$ dbcAmplicons screen -1 ITS_R1.fastq.gz -2 ITS_R2.fastq.gz -O screen/ITS_141219 -R ../refSeqs/ITS_spacers.txt\
\i Output:\
Processed: 100000, PE in ref: 13836, SE in ref: 0\
Processed: 200000, PE in ref: 27638, SE in ref: 0\
Processed: 300000, PE in ref: 41705, SE in ref: 0\
Processed: 400000, PE in ref: 56590, SE in ref: 0\
Processed: 500000, PE in ref: 72206, SE in ref: 0\
Processed: 600000, PE in ref: 88374, SE in ref: 0\
Processed: 700000, PE in ref: 104636, SE in ref: 0\
362350 reads; of these:\
362350 (100.00%) were paired; of these:\
348053 (96.05%) aligned concordantly 0 times\
14066 (3.88%) aligned concordantly exactly 1 time\
231 (0.06%) aligned concordantly >1 times\
----\
348053 pairs aligned concordantly 0 times; of these:\
6088 (1.75%) aligned discordantly 1 time\
----\
341965 pairs aligned 0 times concordantly or discordantly; of these:\
683930 mates make up the pairs; of these:\
568255 (83.09%) aligned 0 times\
31845 (4.66%) aligned exactly 1 time\
83830 (12.26%) aligned >1 times\
21.59% overall alignment rate\
Records processed: 724700, PE in ref: 108888, SE in ref: 0
\i0 \
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\b \cf0 DoubleBarcodeRuns_2019_150807/\
\b0 $ dbcAmplicons screen -1 trn_ab_R1.fastq.gz -2 trn_ab_R2.fastq.gz -O screen/trn_ab
\b _
\b0 150807 -R ../refSeqs/trnT-trnL-trnF.keep\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 Processed: 100000, PE in ref: 2561, SE in ref: 0\
Processed: 200000, PE in ref: 5242, SE in ref: 0\
117131 reads; of these:\
117131 (100.00%) were paired; of these:\
113286 (96.72%) aligned concordantly 0 times\
1590 (1.36%) aligned concordantly exactly 1 time\
2255 (1.93%) aligned concordantly >1 times\
----\
113286 pairs aligned concordantly 0 times; of these:\
865 (0.76%) aligned discordantly 1 time\
----\
112421 pairs aligned 0 times concordantly or discordantly; of these:\
224842 mates make up the pairs; of these:\
223084 (99.22%) aligned 0 times\
481 (0.21%) aligned exactly 1 time\
1277 (0.57%) aligned >1 times\
4.77% overall alignment rate\
Records processed: 234262, PE in ref: 6162, SE in ref: 0\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i0 \cf0 \
$ dbcAmplicons screen -1 trn_cf_R1.fastq.gz -2 trn_cf_R2.fastq.gz -O screen/trn_cd
\b _
\b0 150807 -R ../refSeqs/trnL-trnF.keep\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 Processed: 100000, PE in ref: 712, SE in ref: 0\
Processed: 200000, PE in ref: 1498, SE in ref: 0\
105003 reads; of these:\
105003 (100.00%) were paired; of these:\
103749 (98.81%) aligned concordantly 0 times\
432 (0.41%) aligned concordantly exactly 1 time\
822 (0.78%) aligned concordantly >1 times\
----\
103749 pairs aligned concordantly 0 times; of these:\
21 (0.02%) aligned discordantly 1 time\
----\
103728 pairs aligned 0 times concordantly or discordantly; of these:\
207456 mates make up the pairs; of these:\
207132 (99.84%) aligned 0 times\
200 (0.10%) aligned exactly 1 time\
124 (0.06%) aligned >1 times\
1.37% overall alignment rate\
Records processed: 210006, PE in ref: 1572, SE in ref: 0
\i0 \
\
$ dbcAmplicons screen -1 ITS_R1.fastq.gz -2 ITS_R2.fastq.gz -O screen/ITS
\b _
\b0 150807 -R ../refSeqs/ITS_spacers.txt\
\i 47335 reads; of these:\
47335 (100.00%) were paired; of these:\
43818 (92.57%) aligned concordantly 0 times\
3446 (7.28%) aligned concordantly exactly 1 time\
71 (0.15%) aligned concordantly >1 times\
----\
43818 pairs aligned concordantly 0 times; of these:\
2393 (5.46%) aligned discordantly 1 time\
----\
41425 pairs aligned 0 times concordantly or discordantly; of these:\
82850 mates make up the pairs; of these:\
79135 (95.52%) aligned 0 times\
1610 (1.94%) aligned exactly 1 time\
2105 (2.54%) aligned >1 times\
16.41% overall alignment rate\
Records processed: 94670, PE in ref: 8582, SE in ref: 0
\i0 \
\
$ dbcAmplicons screen -1 rbcL_R1.fastq.gz -2 rbcL_R2.fastq.gz -O screen/rbcL
\b _
\b0 150807 -R ../refSeqs/refGeneSeqPHLAWD.txt\
\i Processed: 100000, PE in ref: 4796, SE in ref: 0\
86303 reads; of these:\
86303 (100.00%) were paired; of these:\
82630 (95.74%) aligned concordantly 0 times\
205 (0.24%) aligned concordantly exactly 1 time\
3468 (4.02%) aligned concordantly >1 times\
----\
82630 pairs aligned concordantly 0 times; of these:\
135 (0.16%) aligned discordantly 1 time\
----\
82495 pairs aligned 0 times concordantly or discordantly; of these:\
164990 mates make up the pairs; of these:\
155701 (94.37%) aligned 0 times\
605 (0.37%) aligned exactly 1 time\
8684 (5.26%) aligned >1 times\
9.79% overall alignment rate\
Records processed: 172606, PE in ref: 8695, SE in ref: 0\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i0 \cf0 \
\
\b DoubleBarcodeRuns_2019_160222/\
\b0 $ dbcAmplicons screen -1 atpB_S1494R_S611_R1.fastq.gz -2 atpB_S1494R_S611_R2.fastq.gz -O screen/atpB_S1494R_S611
\b _
\b0 160222 -R ../refSeqs/refGeneSeqPHLAWD.txt\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 19024 reads; of these:\
19024 (100.00%) were paired; of these:\
17269 (90.77%) aligned concordantly 0 times\
30 (0.16%) aligned concordantly exactly 1 time\
1725 (9.07%) aligned concordantly >1 times\
----\
17269 pairs aligned concordantly 0 times; of these:\
0 (0.00%) aligned discordantly 1 time\
----\
17269 pairs aligned 0 times concordantly or discordantly; of these:\
34538 mates make up the pairs; of these:\
34486 (99.85%) aligned 0 times\
6 (0.02%) aligned exactly 1 time\
46 (0.13%) aligned >1 times\
9.36% overall alignment rate\
Records processed: 38048, PE in ref: 1787, SE in ref: 0
\i0 \
\
$ dbcAmplicons screen -1 atpB_S766R_S3_R1.fastq.gz -2 atpB_S766R_S3_R2.fastq.gz -O screen/atpB_S766R_S3
\b _
\b0 160222 -R ../refSeqs/refGeneSeqPHLAWD.txt\
\i Processed: 100000, PE in ref: 4070, SE in ref: 0\
51256 reads; of these:\
51256 (100.00%) were paired; of these:\
47952 (93.55%) aligned concordantly 0 times\
87 (0.17%) aligned concordantly exactly 1 time\
3217 (6.28%) aligned concordantly >1 times\
----\
47952 pairs aligned concordantly 0 times; of these:\
38 (0.08%) aligned discordantly 1 time\
----\
47914 pairs aligned 0 times concordantly or discordantly; of these:\
95828 mates make up the pairs; of these:\
94367 (98.48%) aligned 0 times\
167 (0.17%) aligned exactly 1 time\
1294 (1.35%) aligned >1 times\
7.95% overall alignment rate\
Records processed: 102512, PE in ref: 4178, SE in ref: 0
\i0 \
\
$ dbcAmplicons screen -1 ITS_R1.fastq.gz -2 ITS_R2.fastq.gz -O screen/ITS
\b _
\b0 160222 -R ../refSeqs/ITS_spacers.txt\
\i 12170 reads; of these:\
12170 (100.00%) were paired; of these:\
10977 (90.20%) aligned concordantly 0 times\
1106 (9.09%) aligned concordantly exactly 1 time\
87 (0.71%) aligned concordantly >1 times\
----\
10977 pairs aligned concordantly 0 times; of these:\
472 (4.30%) aligned discordantly 1 time\
----\
10505 pairs aligned 0 times concordantly or discordantly; of these:\
21010 mates make up the pairs; of these:\
18825 (89.60%) aligned 0 times\
843 (4.01%) aligned exactly 1 time\
1342 (6.39%) aligned >1 times\
22.66% overall alignment rate\
Records processed: 24340, PE in ref: 3283, SE in ref: 0
\i0 \
\
$ dbcAmplicons screen -1 matK_1F_4R_R1.fastq.gz -2 matK_1F_4R_R2.fastq.gz -O screen/matK_1F_4R
\b _
\b0 160222 -R ../refSeqs/refGeneSeqPHLAWD.txt\
\i Processed: 100000, PE in ref: 24912, SE in ref: 0\
Processed: 200000, PE in ref: 50409, SE in ref: 0\
106621 reads; of these:\
106621 (100.00%) were paired; of these:\
54078 (50.72%) aligned concordantly 0 times\
1509 (1.42%) aligned concordantly exactly 1 time\
51034 (47.86%) aligned concordantly >1 times\
----\
54078 pairs aligned concordantly 0 times; of these:\
243 (0.45%) aligned discordantly 1 time\
----\
53835 pairs aligned 0 times concordantly or discordantly; of these:\
107670 mates make up the pairs; of these:\
105894 (98.35%) aligned 0 times\
161 (0.15%) aligned exactly 1 time\
1615 (1.50%) aligned >1 times\
50.34% overall alignment rate\
Records processed: 213242, PE in ref: 53767, SE in ref: 0
\i0 \
\
$ dbcAmplicons screen -1 matK_3F_3R_R1.fastq.gz -2 matK_3F_3R_R2.fastq.gz -O screen/matK_3F_3R
\b _
\b0 160222 -R ../refSeqs/refGeneSeqPHLAWD.txt\
\i Processed: 100000, PE in ref: 7026, SE in ref: 0\
Processed: 200000, PE in ref: 14340, SE in ref: 0\
117064 reads; of these:\
117064 (100.00%) were paired; of these:\
104803 (89.53%) aligned concordantly 0 times\
421 (0.36%) aligned concordantly exactly 1 time\
11840 (10.11%) aligned concordantly >1 times\
----\
104803 pairs aligned concordantly 0 times; of these:\
113 (0.11%) aligned discordantly 1 time\
----\
104690 pairs aligned 0 times concordantly or discordantly; of these:\
209380 mates make up the pairs; of these:\
201086 (96.04%) aligned 0 times\
442 (0.21%) aligned exactly 1 time\
7852 (3.75%) aligned >1 times\
14.11% overall alignment rate\
Records processed: 234128, PE in ref: 16834, SE in ref: 0
\i0 \
\
$ dbcAmplicons screen -1 ndhF_1318_3_R1.fastq.gz -2 ndhF_1318_3_R2.fastq.gz -O screen/ndhF_1318_3
\b _
\b0 160222 -R ../refSeqs/refGeneSeqPHLAWD.txt\
\i Processed: 100000, PE in ref: 1422, SE in ref: 0\
Processed: 200000, PE in ref: 2992, SE in ref: 0\
109843 reads; of these:\
109843 (100.00%) were paired; of these:\
107818 (98.16%) aligned concordantly 0 times\
902 (0.82%) aligned concordantly exactly 1 time\
1123 (1.02%) aligned concordantly >1 times\
----\
107818 pairs aligned concordantly 0 times; of these:\
54 (0.05%) aligned discordantly 1 time\
----\
107764 pairs aligned 0 times concordantly or discordantly; of these:\
215528 mates make up the pairs; of these:\
213980 (99.28%) aligned 0 times\
94 (0.04%) aligned exactly 1 time\
1454 (0.67%) aligned >1 times\
2.60% overall alignment rate\
Records processed: 219686, PE in ref: 3291, SE in ref: 0
\i0 \
\
$ dbcAmplicons screen -1 ndhF_5_803_R1.fastq.gz -2 ndhF_5_803_R2.fastq.gz -O screen/ndhF_5_803
\b _
\b0 160222 -R ../refSeqs/refGeneSeqPHLAWD.txt\
\i Processed: 100000, PE in ref: 2905, SE in ref: 0\
93099 reads; of these:\
93099 (100.00%) were paired; of these:\
88364 (94.91%) aligned concordantly 0 times\
339 (0.36%) aligned concordantly exactly 1 time\
4396 (4.72%) aligned concordantly >1 times\
----\
88364 pairs aligned concordantly 0 times; of these:\
128 (0.14%) aligned discordantly 1 time\
----\
88236 pairs aligned 0 times concordantly or discordantly; of these:\
176472 mates make up the pairs; of these:\
175407 (99.40%) aligned 0 times\
557 (0.32%) aligned exactly 1 time\
508 (0.29%) aligned >1 times\
5.80% overall alignment rate\
Records processed: 186198, PE in ref: 5674, SE in ref: 0
\i0 \
\
$ dbcAmplicons screen -1 ndhF_803_1318_R1.fastq.gz -2 ndhF_803_1318_R2.fastq.gz -O screen/ndhF_803_1318
\b _
\b0 160222 -R ../refSeqs/refGeneSeqPHLAWD.txt\
\i Processed: 100000, PE in ref: 17733, SE in ref: 0\
Processed: 200000, PE in ref: 35986, SE in ref: 0\
113408 reads; of these:\
113408 (100.00%) were paired; of these:\
74048 (65.29%) aligned concordantly 0 times\
3632 (3.20%) aligned concordantly exactly 1 time\
35728 (31.50%) aligned concordantly >1 times\
----\
74048 pairs aligned concordantly 0 times; of these:\
603 (0.81%) aligned discordantly 1 time\
----\
73445 pairs aligned 0 times concordantly or discordantly; of these:\
146890 mates make up the pairs; of these:\
145195 (98.85%) aligned 0 times\
371 (0.25%) aligned exactly 1 time\
1324 (0.90%) aligned >1 times\
35.99% overall alignment rate\
Records processed: 226816, PE in ref: 40966, SE in ref: 0
\i0 \
\
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\b \cf0 3_reduce : reduce amplicons to a consensus sequence (or ambiguous, or ID alleles) \
\b0 \
\b DoubleBarcodeRuns_2019_141219/screen/\
\b0 $ reduce_amplicons.R -o reduced/trn_cf_141219 -p consensus trn_cd_141219.mapped \
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 Output: \
Reads total: 34253\
Reads discarded: 517 (1.51%)\
Reads combined: 4917 (14.57%)\
combined extended: 1718 (34.94%)\
combined short: 3199 (65.06%)
\i0 \
\
$ reduce_amplicons.R -o reduced/ITS_141219 -p consensus ITS_141219.mapped\
\i Output:\
Reads total: 108888\
Reads discarded: 3466 (3.18%)\
Reads combined: 21207 (20.12%)\
combined extended: 3406 (16.06%)\
combined short: 17801 (83.94%)
\i0 \
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\b \cf0 DoubleBarcodeRuns_2019_150807/screen/\
\b0 $ reduce_amplicons.R -o reduced/trn_ab_150807 -p consensus trn_ab_150807.mapped\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 Reads total: 6162\
Reads discarded: 351 (5.7%)\
Reads combined: 4131 (71.09%)\
combined extended: 2754 (66.67%)\
combined short: 1377 (33.33%)
\i0 \
\
$ reduce_amplicons.R -o reduced/trn_cf_150807 -p consensus trn_cd_150807.mapped\
\i Reads total: 1572\
Reads discarded: 199 (12.66%)\
Reads combined: 941 (68.54%)\
combined extended: 26 (2.76%)\
combined short: 915 (97.24%)
\i0 \
\
\i $ reduce_amplicons.R -o reduced/trn_cf_150807 -p consensus trn_cd_150807.mapped -f 0.001 -s 3\
Reads total: 1572\
Reads discarded: 199 (12.66%)\
Reads combined: 941 (68.54%)\
combined extended: 26 (2.76%)\
combined short: 915 (97.24%)\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i0 \cf0 \
$ reduce_amplicons.R -o reduced/ITS_150807 -p consensus ITS_150807.mapped\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 Reads total: 8582\
Reads discarded: 1183 (13.78%)\
Reads combined: 6181 (83.54%)\
combined extended: 562 (9.09%)\
combined short: 5619 (90.91%)
\i0 \
\
$ reduce_amplicons.R -o reduced/rbcL_150807 -p consensus rbcL_150807.mapped\
\i Reads total: 8695\
Reads discarded: 847 (9.74%)\
Reads combined: 6230 (79.38%)\
combined extended: 59 (0.95%)\
combined short: 6171 (99.05%)
\i0 \
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\b \cf0 DoubleBarcodeRuns_2019_160222/screen/\
\b0 $ reduce_amplicons.R -o reduced/atpB_S1494R_S611_160222 -p consensus atpB_S1494R_S611_160222.mapped\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 Reads total: 1787\
Reads discarded: 18 (1.01%)\
Reads combined: 21 (1.19%)\
combined extended: 2 (9.52%)\
combined short: 19 (90.48%)
\i0 \
\
$ reduce_amplicons.R -o reduced/atpB_S766R_S3_160222 -p consensus atpB_S766R_S3_160222.mapped\
\i Reads total: 4178\
Reads discarded: 280 (6.7%)\
Reads combined: 799 (20.5%)\
combined extended: 48 (6.01%)\
combined short: 751 (93.99%)
\i0 \
\
$ reduce_amplicons.R -o reduced/ITS_160222 -p consensus ITS_160222.mapped\
\i Reads total: 3283\
Reads discarded: 323 (9.84%)\
Reads combined: 1757 (59.36%)\
combined extended: 191 (10.87%)\
combined short: 1566 (89.13%)
\i0 \
\
$ reduce_amplicons.R -o reduced/matK_1F_4R_160222 -p consensus matK_1F_4R_160222.mapped\
\i Reads total: 53767\
Reads discarded: 164 (0.31%)\
Reads combined: 46354 (86.48%)\
combined extended: 44772 (96.59%)\
combined short: 1582 (3.41%)\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i0 \cf0 \
$ reduce_amplicons.R -o reduced/matK_3F_3R_160222 -p consensus matK_3F_3R_160222.mapped\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 Reads total: 16834\
Reads discarded: 667 (3.96%)\
Reads combined: 5627 (34.81%)\
combined extended: 1265 (22.48%)\
combined short: 4362 (77.52%)
\i0 \
\
$ reduce_amplicons.R -o reduced/ndhF_1318_3_160222 -p consensus ndhF_1318_3_160222.mapped\
\i Reads total: 3291\
Reads discarded: 118 (3.59%)\
Reads combined: 1102 (34.73%)\
combined extended: 39 (3.54%)\
combined short: 1063 (96.46%)\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i0 \cf0 \
$ reduce_amplicons.R -o reduced/ndhF_5_803_160222 -p consensus ndhF_5_803_160222.mapped\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 Reads total: 5674\
Reads discarded: 385 (6.79%)\
Reads combined: 767 (14.5%)\
combined extended: 42 (5.48%)\
combined short: 725 (94.52%)\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i0 \cf0 \
$ reduce_amplicons.R -o reduced/ndhF_803_1318_160222 -p consensus ndhF_803_1318_160222.mapped\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 Reads total: 40966\
Reads discarded: 129 (0.31%)\
Reads combined: 40426 (98.99%)\
combined extended: 38265 (94.65%)\
combined short: 2161 (5.35%)\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i0 \cf0 \
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i \cf0 OUTPUT INTERPRETATION:
\i0 consensus.reduced.fasta
\b \
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\b0 \cf0 >Heuchera_parvifolia_var_dissecta_14449:trnLF|merged|1|6|9|0.667|0.021\
species name | merged or read 1/2 | number of alleles (occurrence only) | number of reads contributing to consensus | total number of reads | percent reads in consensus | variability of total number of reads \
\
\
\b ##### copy reduced consensus to local HD: Sawtooth-Alpine-PD/output/00_MiSeq/3_reduce\
\b0 scp -r brlab@141.211.236.50:~/Documents/hmarx/Sawtooth/seqs/DoubleBarcodeRuns_2019_141219/screen/reduced/* 141219/\
\
scp -r brlab@141.211.236.50:~/Documents/hmarx/Sawtooth/seqs/DoubleBarcodeRuns_2019_150807/screen/reduced/* 150807/\
\
scp -r brlab@141.211.236.50:~/Documents/hmarx/Sawtooth/seqs/DoubleBarcodeRuns_2019_160222/screen/reduced/* 160222/\
\
\
\b 4_concensus: combine gene regions across runs to one fasta\
\b0 cat ../3_reduce/141219/ITS_141219/consensus.reduced.fasta ../3_reduce/150807/ITS_150807/consensus.reduced.fasta ../3_reduce/160222/ITS_160222/consensus.reduced.fasta > 01_separateReads/ITS.fasta\
\
cat ../3_reduce/141219/trn_cf_141219/consensus.reduced.fasta ../3_reduce/150807/trn_cf_150807/consensus.reduced.fasta > 01_separateReads/trn_cf.fasta\
\
\b 4_concensus/01_separateReads:
\b0 Separate reads for each gene region\
read1 \'97> ITS.read1\
read 2 \'97> ITS.read2\
merged \'97> ITS.merged\
\
* copy consensus.reduced.fasta and rename to gene region:\
atpB_S1494R_S611_160222 = atpB1\
atpB_S766R_S3_160222 = atpB2\
\
matK_1F_4R_160222 = matK1\
matK_3F_3R_160222 = matk2\
\
ndhF_5_803_160222 = ndhF1\
ndhF_803_1318_160222 = ndhF2\
ndhF_1318_3_160222 = ndhF3\
\
***COPY AND PAST TO NEW FILE\
\
* Add a < at the end of header\
find: ^(>.+)\
replace: \\1<\
\
* Remove returns from end of all lines\
find: \\r\
replace: nothing\
\
* add one return back for each seq\
find: >\
replace: \\r>\
\
* add one return back after header \
find: <\
replace: \\r\
\
***COPY AND PAST TO NEW FILE\
\
* Remove all read2\
^(>.+read2.+\\r.+)\
\\r\
*Remove all merged \
^(>.+merged.+\\r.+)\
\\r\
\
* remove space at beginning of lines\
^\\s\
\
***SAVE as .read1\
\
* Remove all read1 \
^(>.+read1.+\\r.+)\
\\r\
\
*Remove all merged \
^(>.+merged.+\\r.+)\
\\r\
\
* remove space at beginning of lines\
^\\s\
\
***SAVE as .read2\
\
*Remove all merged \
\
^(>.+merged.+\\r.+)\
\\r\
\
* remove space at beginning of lines\
^\\s\
\
* remove last line \
\\r\\Z\
\\Z\
\
\
\
\b 4_concensus/02_concatGeneSegments\
\
rbcL:\
\b0 1) concatenate read1 and read2 for each gene region
\b \
\b0 * copy read1 and read2 this folder\
* remove | to line return (keep gene region for now)\
find: (\\|+.+\\r)\
replace: \\r\
* get rid of colon from read names\
$ for i in *.fasta; do sed -i \'93\'94 \'92s/:/_/g\'92 $i;done \
* prepare a concat file:\
$ ls > names.concat.txt\
* edit in TextWrangler: \
Find: (^.+)\\r(.+)\
Replace:phyutility -concat -in \\1 \\2 -out \\1\
\
Search:read1.fasta$\
Replace:R1_R2.nex\
\
$ phyutility -concat -in rbcL.read1.fasta rbcL.read2.fasta -out rbcL.R1_R2.nex\
\
Save as .fasta \
\b \
\b0 \
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\i\b \cf0 For the more complex regions:
\i0\b0 \
\
Copy reads from /01_separateReads into Geneious\
Align each segment;\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f2 \cf0 (for atpB, rev. Complement)
\f0 \
Copy alignment, and modify: correct reading frame to 1, Trimmed ends so all sequences in segment are equal length;\
Mapped reads from each segment to reference to make sure non-overlapping segments, if so, remove (
\f2 atpB1_r2
\f0 , );\
\
Manually removed segments with bad assemblies in GenBank (listed below for each gene region)\
\f2 \
Export alignment as whole: gene.GenBankReference.fasta (e.g. atpB.HQ384707.fasta)\
\
Remove reference & Separate gene regions:\
atpB1_R1.HQ384707.fasta\
atpB1_R2.HQ384707.fasta\
atpB2_R1.HQ384707.fasta\
atpB2_R2.HQ384707.fasta\
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f0 \cf0 Textwranger:
\f2 \
** remove gaps from each\
\f0 ** remove header:\
Find: (\\:+.+\\r)\
Rep: \\r\
\
\
\b atpB:
\b0 \
atpB2_R2:\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f2 \cf0 Gentiana_calycosa_14414\
Castilleja_miniata_14369\
Phyllodoce_empetriformis_14359\
Penstemon_montanus_var_idahoensis_14279\
Penstemon_montanus_var_idahoensis_13570\
Bupleurum_americanum_14364\
Penstemon_montanus_var_idahoensis_14234\
Penstemon_procerus_var_formosus_14447\
Heuchera_parvifolia_14449\
Chionophila_tweedyi_14294\
Phyllodoce_glanduliflora_14023\
Sedum_debile_14272\
Erythranthe_tilingii_14413\
Parnassia_fimbriata_14370\
Cassiope_mertensiana_var_gracilis_14292\
Phyllodoce_empetriformis_14219\
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f0 \cf0 atpB2_R1:
\f2 \
Sedum_debile_14272\
\
\f0 atpB1_R2:\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f2 \cf0 Carex_straminiformis_14226\
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f0\b \cf0 matK: \
\b0 Removed merged\
Read1:\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f2 \cf0 Viola_cf_adunca_14576\
Erigeron_compositus_14623\
Androsace_septentrionalis_14464\
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\b \cf0 ndhF:\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\b0 \cf0 Aligned segments in Geneious, trimmed, and exported as .fasta\
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\b \cf0 trnTLF:\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\b0 \cf0 Aligned segments in Geneious, trimmed, and exported as .fasta\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f0\b \cf0 \
ITS:
\b0 \
Aligned with R1, R2 MUSCLE in Geneious, exported as .fasta\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f2 \cf0 ***REMOVED NA (neg) seqs x 2\
Discard *merged\
\
\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f0\b \cf0 4_concensus/02_concatGeneSegments:
\b0 merge MiSeq segments for each region\
* concatenate regions with multiple segments with phyutility\
* export .fasta from Geneious\
\
\b rbcL:
\b0 one segment only\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f2 \cf0 $ mafft --reorder rbcL.R1_R2.fasta > rbcL.R1_R2.aln.fasta\
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f0 \cf0 \
\b atpB:
\b0 two
\b
\b0 overlapping segments \
mafft --reorder atpB2_R2.HQ384707.fasta > atpB2_R2.HQ384707.aln.fasta\
mafft --reorder atpB2_R1.HQ384707.fasta > atpB2_R1.HQ384707.aln.fasta\
mafft --reorder atpB1_R2.HQ384707.fasta > atpB1_R2.HQ384707.aln.fasta\
mafft --reorder atpB1_R1.HQ384707.fasta > atpB1_R1.HQ384707.aln.fasta\
phyutility -concat -in atpB2_R2.HQ384707.aln.fasta atpB2_R1.HQ384707.aln.fasta atpB1_R2.HQ384707.aln.fasta atpB1_R1.HQ384707.aln.fasta -out atpB.miseq.aln.nex\
* export .fasta from Geneious \'97> atpB.miseq.aln.fasta
\b \
\
matK:
\b0 two segments with gap\
$ mafft --reorder matK1_R1.KF907876.fasta > matK1_R1.KF907876.aln.fasta\
$ mafft --reorder matK1_R2.KF907876.fasta > matK1_R2.KF907876.aln.fasta\
$ mafft --reorder matK2_R1.KF907876.fasta > matK2_R1.KF907876.aln.fasta\
$ mafft --reorder matK2_R2.KF907876.fasta > matK2_R2.KF907876.aln.fasta\
$ phyutility -concat -in matK1_R1.KF907876.aln.fasta matK1_R2.KF907876.aln.fasta matK2_R1.KF907876.aln.fasta matK2_R2.KF907876.aln.fasta -out matK.miseq.aln.nex
\b \
\b0 * save as .fasta from Geneious, cleaned up a but \'97> matK.miseq.aln.mod.fasta
\b \
\