BACE, Curto - PCR

Performed PCR:

	1 Rxn (μl)	Rxn Number	Total
		96
dH2O	11.3		1084.8
Premix F	15		1440.0
pA (50μM)	0.2		19.2
pH' (50μM)	0.2		19.2
Taq (5units/μl)	0.3		28.8
DNA	3.0

rDNA 16S Gene for 1500 bp 

Primers Used:

Forward Primer - pA -> 27f / E8F

Reverse Primer - pH' -> 1525f / E1541F

ThermoCycler:
95 for 4:00 min
*95 for 0:40 min
*55.5 for 0:30 min
*72 for 2 min
72 for 3:30 min
Hold at 4
*Repeat for 30 cycles

LOOKED REALLY GOOD!

Transfer 20 uL to new plate - READY FOR SEQ

Claudia sent out to Beckman Genomics Institute on 1/13/15

Curto - PCR

Took Kristen's Curtobacterium strains from deep freeze and plated.

Performed PCR:

	1 Rxn (μl)	Rxn Number	Total
		12
dH2O	13.3		159.6
Premix F	15		180
pA (50μM)	0.2		2.4
pH' (50μM)	0.2		2.4
Taq (5units/μl)	0.3		3.6
DNA	1

rDNA 16S Gene for 1500 bp 

Primers Used:

Forward Primer - pA -> 27f / E8F

Reverse Primer - pH' -> 1525f / E1541F

5 of the 9 strains amplified

Need to reevaluate strains that failed

EMP Biom Files Pt. VII

Went back to the hdf5 file and attempted to solve problem in MatLab (Rich helped a lot!)

Solved the array issue:

%% Load the data
a=h5info('name of hdf file');% return structured array of the hdf
% hieracrchy for reference
observdata=h5read('name of hdf file','/observation/matrix/data');
observIndices=h5read('name of hdf file','/observation/matrix/indices');
observIndptr=h5read('name of hdf file','/observation/matrix/indptr');
ids=h5read('name of hdf file','/observation/ids');

%% Get the OTU indices
% You need a cell array of strings of your desired OTUs stored as variable
% qList

for i=1:length(qList)
    qInd=find(strcmp(qList,ids));
end

%% find the data

outmat=zeros(length(qList),length(sampleIndptr));

for i=1:length(qInd)
    p=observdata(observIndptr(qInd(i)):observIndptr(qInd(i)+1));
    pI=observIndices(observIndptr(qInd(i)):observIndptr(qInd(i)+1));
    for j=1:length(pI)
        outmat(i,pI(j)+1)=p(j); %plus one to correct for matlab python coordinate changes
    end
end

Merged files and got the following with all metadata!!!!!

EMP Biom Files Pt. VI - Green Planet

Got access to the Green Planet server and am able to login.

Was informed by Chad Cantwell that QIIME may or may not be installed in the new environment (server was updated recently and it may not be running)

Contacted  Steve Hatosy (from Adam's lab) and was super helpful.
When you login to the server (through ssh), type: . /sopt/qiime/set_paths_1.4
This should set the paths to the QIIME script

Code to run:
split_otu_table_by_taxonomy.py
-i full_emp_table_w_tax.biom #open source biom file
-L 5 #split taxonomy at Family level
-o ./L5/ #file directory

>> SystemError: Negative size passed to PyString_FromStringAndSize

This is (I think) due to large size of the input file (biom file 2.63 GB). From my correspondence with people in the Knight Lab, the file should need ~30GB of memory to load.

Protocol for Cluster:

1. ssh -Y abchase@gplogin3.ps.uci.edu
2. enter login information
3. pwd #gets cluster directory 
4. exit cluster
5. from local terminal. you can upload files to the cluster:
1. scp /Users/MartinyLab/Desktop/alexs-stuff/EMP/EMPopen/full_emp_table_w_tax.biom abchase@gplogin1.ps.uci.edu:/home/abchase
6. Run QIIME code with cluster file directory

BACE - Isolate Strains

12/8/2014
Got done streaking the cultures grown on BACE media (n=39). Took no cultures from 1:10000 dilutions

12/9/2014
Got done streaking the cultures grown on Loma Ridge media (n=40). Took no cultures from 1:10000 dilutions

Curto - Kristen's Isolates

Need to find Kristen's Curtobacterium isolates. Jen sent spreadsheet with isolates that have been deep frozen "~/Documents/Research/m.curtobacterium/bacterial-strains/bacterial-cultures.xlsx"

From Lab Intranet, strains to consider (n=9):

109
115
136
145
163
171
183*
190*
213**

*originally classified as Okibacterium in Kristen's spreadsheet
**not listed in spreadsheet

Kristen says the spreadsheet in outdated but Intranet should be correct.

Streaked strains on to new LB plates and, eventually, sequence 16S to verify that they are Curtobacterium

BACE - Culture litter

1. Took Boston litter and ran through autoclaved sieves (2000 um -> 250 um -> 25 um)
1. ***NEXT TIME - GRIND UP LITTER BEFOREHAND
2. Scraped water and residue in bottom two filters into 50mL conicals
3. Add litter residue to vacuum filter (no vacuum attached) with 100 um filter
4. Rinse conicals 2x with autoclaved DI H2O
5. Total of 200 mL of litter residue and add to new set of 50mL conicals
6. Dilute each tube to 1x, 1:100, 1:1000, 1:10000
7. Plate 100 uL of each dilution on media plates:
1. For this experiment, plated on both Loma Ridge Media and BACE Media

EMP Biom Files Pt. V

Still working on open source files. not really sure how to access array data from hdf5 files

However, wrote short code to organize the 2 OTUs from the closed_ref_emp_table to combine with merged mapping file:

import os
import csv

mydir = os.path.expanduser("~/Desktop/alexs-stuff/EMP/")


in_file = mydir + "EMP_10k_merged_mapping_final.txt" #master mapping file

#need txt file with sample ids that had curto hits
wanted_file = mydir + "EMPclosed/sample-ids-curto.txt" 

out_file = mydir + "EMPclosed/curto-samples.csv"

wanted = set()

with open(wanted_file) as f:
for line in f:
line = line.strip()
if line != "":
wanted.add(line)

count = 0

with open(in_file, "rb") as tsvin, open(out_file, "wb") as csvout:
tsvin = csv.reader(tsvin, delimiter = '\t')
csvout = csv.writer(csvout)

for row in tsvin:

if row[0] in wanted:
count = count + 1
csvout.writerows([row])


print "Converted %i records" % count

Output looks like this in excel after some editing:
Total samples: 136 with 111 in the merged mapping file