brew install https://raw.github.com/Homebrew/homebrew-science/master/fastx_toolkit.rb

brew link --overwrite fastx_toolkit

Linking /usr/local/Cellar/fastx_toolkit/0.0.14... 

Error: Could not symlink include/gtextutils/gtextutils/container_join.h

/usr/local/include/gtextutils/gtextutils is not writable.

brew install https://raw.github.com/Homebrew/homebrew-science/master/fastx_toolkit.rb

######################################################################## 100.0%

Warning: fastx_toolkit-0.0.14 already installed, it's just not linked

sudo chown -R laurelyohe /usr/local/include

brew link --overwrite fastx_toolkit

Alright! I think it is installed. I have avoided using fastx-toolkit because it is such a pain in the butt to install, but alas, the uncertain has become certain. Why am I doing this? Because I am getting these alarming warning messages while Trinity is running. It seems to carry on regardless but it doesn't seem right.

...

warning: ignoring read XXXX since it cannot decipher if /1 or /2 of a pair.

Error: note that there were 4329123 reads that could not be deciphered as being /1 or /2 of a PE fragment. Hopefully, these were SE reads that should have been ignored. Otherwise, please research this further.

Okay, awesome. Seems like someone else ran into this problem:

http://sourceforge.net/p/trinityrnaseq/mailman/message/31448854/

After looking at what my input files were before and after trimming, it is clear that the fastq files have lost track of being from the right or left pair. Uggh! I knew that girl's Trinity help was too good to be true.

head Arjam_MOE_R1.fq

@HWI-ST885:197:HA2RPADXX:1:1101:1464:2292 1:N:0:ACAGTG

GCCCTTGCCGCCAGCTGGCGCGGCCACGCAGCGGCTGAGCGAGTCTAGGCGCGCGCTGTACTCGGTGAACTTCTTTTGGTAGCGCAGAGCAGTCATTTGC

+

CCCFFFFFHHHHHJJJIJJJJJJJJJJJJJJIHHFDDCDDDDDDDDDDDCBBDBBBDDDDDEDDD@DDDDDDDDDDDDDACCDDDDDDDDDDDDDDEEED

@HWI-ST885:197:HA2RPADXX:1:1101:1438:2386 1:N:0:ACAGTG

GGGTGGAGCAAATTCTGGGCCAAAAGTAGTAGGTTCTGAAGGAGGAGGCAATGGTGGTGGTGGTGGACGTGGTCTCCTGAAGTGTCTGCGACTCTCACCA

+

CCCFFFFFDHHHHJGGHGIGIJJJHIFHIGIIJJJIIIIGHGGHI?FGHIGIIJ8@=5BEHHH9BEFEDCDDBDDDDDDCCD>@CCDDDDBDBBDDCCBC

@HWI-ST885:197:HA2RPADXX:1:1101:1310:2396 1:N:0:ACAGTG

CTTGGGTCGTGAGTGAGAACAGGCTGGTAGACGGGGCGCTCGCCGAAGGCTGGGATGAAGTCCCGAAACTAAACCCACCAGCGCTGGATGAGGAGAAAGA

head Arjam_MOE_R2.fq

@HWI-ST885:197:HA2RPADXX:1:1101:1464:2292 2:N:0:ACAGTG

CAACGGCGCGCTGCAGGCCCGGCTCGCCGCGCTGCACAAGGCTTTCAAGAAGGAGGCTCTGCGCGCAGGCAAGCGCGAGAAGTCGCTGGTGGCGCAGCTG

+

CCCFFFFFHHHHHJJJJJJIIJIEIBEHBBBBBDDD<CDDDDDDDCCDDDDDD?ABBBDDDDDDDBBDD@DDDDDDD<BBDDDDDDDBB<CBABD@BDDC

@HWI-ST885:197:HA2RPADXX:1:1101:1438:2386 2:N:0:ACAGTG

CCTGTTTCTCGCCTGGTGAGAGTCGCAGACACTTCAGGAGACCACGTCCACCACCACCACCATTGCCTCCTCCTTCAGAACCTACTACTTTTGGCCCAGA

+

BBBFFFFFHHGHHIJJGHHHHIGHIIIIJJJJJJJJJJIJIJFJJGHIIHJGHJJEHHHFFFFEEEEEEDDDDDDDDDDDDDDDDDDDDDEDDDDDDBDD

@HWI-ST885:197:HA2RPADXX:1:1101:1310:2396 2:N:0:ACAGTG

CAGGCGGGTTCACGTTTGGCACCGCAAAGACGGCGACAACCACCCCTGCCACCGGCTTTTCTTTCTCCTCATCCAGCGCTGGTGGGTTTAGTTTCGGGAC

Okay see how there is a 1 and a 2 in the title line?

head Arjam_MOE_R1_trim.fq

@HWI-ST885:197:HA2RPADXX:1:1101:1464:2292

GCCCTTGCCGCCAGCTGGCGCGGCCACGCAGCGGCTGAGCGAGTCTAGGCGCGCGCTGTACTCGGTGAACTTCTTTTGGTAGCGCAGAGCAGTCATTTGC

+

CCCFFFFFHHHHHJJJIJJJJJJJJJJJJJJIHHFDDCDDDDDDDDDDDCBBDBBBDDDDDEDDD@DDDDDDDDDDDDDACCDDDDDDDDDDDDDDEEED

@HWI-ST885:197:HA2RPADXX:1:1101:1438:2386

GGGTGGAGCAAATTCTGGGCCAAAAGTAGTAGGTTCTGAAGGAGGAGGCAATGGTGGTGGTGGTGGACGTGGTCTCCTGAAGTGTCTGCGACTCTCACCA

+

CCCFFFFFDHHHHJGGHGIGIJJJHIFHIGIIJJJIIIIGHGGHI?FGHIGIIJ8@=5BEHHH9BEFEDCDDBDDDDDDCCD>@CCDDDDBDBBDDCCBC

@HWI-ST885:197:HA2RPADXX:1:1101:1310:2396

CTTGGGTCGTGAGTGAGAACAGGCTGGTAGACGGGGCGCTCGCCGAAGGCTGGGATGAAGTCCCGAAACTAAACCCACCAGCGCTGGATGAGGAGAAAGA

head Arjam_MOE_R2_trim.fq

@HWI-ST885:197:HA2RPADXX:1:1101:1464:2292

CAACGGCGCGCTGCAGGCCCGGCTCGCCGCGCTGCACAAGGCTTTCAAGAAGGAGGCTCTGCGCGCAGGCAAGCGCGAGAAGTCGCTGGTGGCGCAGCTG

+

CCCFFFFFHHHHHJJJJJJIIJIEIBEHBBBBBDDD<CDDDDDDDCCDDDDDD?ABBBDDDDDDDBBDD@DDDDDDD<BBDDDDDDDBB<CBABD@BDDC

@HWI-ST885:197:HA2RPADXX:1:1101:1438:2386

CCTGTTTCTCGCCTGGTGAGAGTCGCAGACACTTCAGGAGACCACGTCCACCACCACCACCATTGCCTCCTCCTTCAGAACCTACTACTTTTGGCCCAGA

+

BBBFFFFFHHGHHIJJGHHHHIGHIIIIJJJJJJJJJJIJIJFJJGHIIHJGHJJEHHHFFFFEEEEEEDDDDDDDDDDDDDDDDDDDDDEDDDDDDBDD

@HWI-ST885:197:HA2RPADXX:1:1101:1310:2396

CAGGCGGGTTCACGTTTGGCACCGCAAAGACGGCGACAACCACCCCTGCCACCGGCTTTTCTTTCTCCTCATCCAGCGCTGGTGGGTTTAGTTTCGGGAC

See how it goes away? Bollocks! This happens after running python ~/Scripts/q-trim.py.

Let's see if this strips it.

fastq_quality_filter -i Arjam_MOE_R1.fq -o Arjam_MOE_R1_fastxtrimmed.fastq -q 20 -p 80 -Q 33 -v

Quality cut-off: 20

Minimum percentage: 80

Input: 15149790 reads.

Output: 14691119 reads.

head Arjam_MOE_R1_fastxtrimmed.fastq 

@HWI-ST885:197:HA2RPADXX:1:1101:1464:2292 1:N:0:ACAGTG

GCCCTTGCCGCCAGCTGGCGCGGCCACGCAGCGGCTGAGCGAGTCTAGGCGCGCGCTGTACTCGGTGAACTTCTTTTGGTAGCGCAGAGCAGTCATTTGC

+

CCCFFFFFHHHHHJJJIJJJJJJJJJJJJJJIHHFDDCDDDDDDDDDDDCBBDBBBDDDDDEDDD@DDDDDDDDDDDDDACCDDDDDDDDDDDDDDEEED

@HWI-ST885:197:HA2RPADXX:1:1101:1438:2386 1:N:0:ACAGTG

GGGTGGAGCAAATTCTGGGCCAAAAGTAGTAGGTTCTGAAGGAGGAGGCAATGGTGGTGGTGGTGGACGTGGTCTCCTGAAGTGTCTGCGACTCTCACCA

+

CCCFFFFFDHHHHJGGHGIGIJJJHIFHIGIIJJJIIIIGHGGHI?FGHIGIIJ8@=5BEHHH9BEFEDCDDBDDDDDDCCD>@CCDDDDBDBBDDCCBC

@HWI-ST885:197:HA2RPADXX:1:1101:1310:2396 1:N:0:ACAGTG

CTTGGGTCGTGAGTGAGAACAGGCTGGTAGACGGGGCGCTCGCCGAAGGCTGGGATGAAGTCCCGAAACTAAACCCACCAGCGCTGGATGAGGAGAAAGA

Cool! Looks like it kept the extended header on.

fastq_quality_filter -i Arjam_MOE_R2.fq -o Arjam_MOE_R2_fastxtrimmed.fastq -q 20 -p 80 -Q 33 -v

Quality cut-off: 20

Minimum percentage: 80

Input: 15149790 reads.

Output: 14464395 reads.

discarded 685395 (4%) low-quality reads.

For once something worked. Oh yeah, just as a side note if you have illumina reads you must put -Q 33 parameter. This of course, is an undocumented fix discovered by users. Three+ years after this discovery, -Q remains to be documented in the fastx-toolkit. 

I still am going to do the pair matching before running trinity again.

~/Scripts/both.py Arjam_MOE_R1_fastxtrimmed.fastq Arjam_MOE_R2_fastxtrimmed.fastq 

Trinity.pl --seqType fq --left Arjam_MOE_R1_fastxtrimmed.fastq.both --right Arjam_MOE_R2_fastxtrimmed.fastq.both --CPU 4 --JM 20G --output output_4

----------------------------------------------------------------

Yay! I fixed the /1 /2 problem. You can see that the fasta files trinity creates there is now the /1 and /2. Now we will see if we can get past chrysalis and if the other error with the c++libs is fixed.

head left.fa
>HWI-ST885:197:HA2RPADXX:1:1101:1464:2292/1
GCCCTTGCCGCCAGCTGGCGCGGCCACGCAGCGGCTGAGCGAGTCTAGGCGCGCGCTGTACTCGGTGAACTTCTTTTGGTAGCGCAGAGCAGTCATTTGC
>HWI-ST885:197:HA2RPADXX:1:1101:1438:2386/1
GGGTGGAGCAAATTCTGGGCCAAAAGTAGTAGGTTCTGAAGGAGGAGGCAATGGTGGTGGTGGTGGACGTGGTCTCCTGAAGTGTCTGCGACTCTCACCA
>HWI-ST885:197:HA2RPADXX:1:1101:1310:2396/1
CTTGGGTCGTGAGTGAGAACAGGCTGGTAGACGGGGCGCTCGCCGAAGGCTGGGATGAAGTCCCGAAACTAAACCCACCAGCGCTGGATGAGGAGAAAGA
>HWI-ST885:197:HA2RPADXX:1:1101:1346:2468/1
ATGGAGCCCAGGCCTCCAGTGCAGAGTGAGTGCTTCCTTCCATGGTCCCCATGCCATCGTGTGACAAGTTCTGTGACCTGATTTCCAGCACTGTCATCCA
>HWI-ST885:197:HA2RPADXX:1:1101:1467:2481/1
GGCCCAGGGAGTCCTCCACCACCCCCTCCCCTTTCCTGGCCTGCTCTCTAATTCTCTAGAAACCTTCCTGTGTATCCTGCCTACTTAAACCCTGCATCCC
head right.fa
>HWI-ST885:197:HA2RPADXX:1:1101:1464:2292/2
CAACGGCGCGCTGCAGGCCCGGCTCGCCGCGCTGCACAAGGCTTTCAAGAAGGAGGCTCTGCGCGCAGGCAAGCGCGAGAAGTCGCTGGTGGCGCAGCTG
>HWI-ST885:197:HA2RPADXX:1:1101:1438:2386/2
CCTGTTTCTCGCCTGGTGAGAGTCGCAGACACTTCAGGAGACCACGTCCACCACCACCACCATTGCCTCCTCCTTCAGAACCTACTACTTTTGGCCCAGA
>HWI-ST885:197:HA2RPADXX:1:1101:1310:2396/2
CAGGCGGGTTCACGTTTGGCACCGCAAAGACGGCGACAACCACCCCTGCCACCGGCTTTTCTTTCTCCTCATCCAGCGCTGGTGGGTTTAGTTTCGGGAC
>HWI-ST885:197:HA2RPADXX:1:1101:1346:2468/2
GGGGGTGGAAATTTTGAGACAAATGTTGATTCTTGGTGAGTTGATGAGTCTTTTCTAGACACATAGAGAAGGTGCTGAAGATTGAGAGAAAACGCCTTCC
>HWI-ST885:197:HA2RPADXX:1:1101:1467:2481/2

GTCAAACTCGTTGTAGCAGATTCTACTTGGGATGCAGGGTTTAAGTAGGCAGGATACACAGGAAGGTTTCTAGAGAATTAGAGAGCAGGCCAGGAAAGGG

To be continued.

Okay we have FINALLY updated our operating system.

Apparently Mavericks already has commandline tools installed so it is not necessary to install again. However, there are some issues if you install Homebrew and the gcc compiler not being linked. So starting from scratch, here is what do do.

#install homebrew

ruby -e "$(curl -fsSL https://raw.github.com/Homebrew/homebrew/go/install)"

#check to see your comp is ready to brew

brew doctor

#i had several errors/warnings (errors indicated in orange)

/usr/bin comes before /usr/local/bin

export PATH='/usr/local/bin:$PATH' >> ~/.bash_profile

/usr/local/lib/gcc is not writable.

sudo chown -R laurelyohe /usr/local

brew install homebrew/science/bowtie

brew install apple-gcc42

brew install samtools

brew install trinity

Okay, everything seems good to go. Now I am unzipping the transcriptome output from trimmomatic. 

Because I did such a horrid job taking comprehensible notes while in London, I am going to be more efficient this time around with transcriptome assembly.

We have our illumina RNA-seq reads back (yay!).

To download the reads from UArizona, you have to download this horridly complicated program called irods (http://uagc.arl.arizona.edu/resources/user-guide). After much struggle, the easiest way to get things installed and compiled is the "non-fancy" way. HOWEVER, you MUST download the most recent version of iRods (as of right now 3.3.1)

Navigate to your iRODs folder. Things in blue are what you type. Things in pink are what you would change differently based on your computer.

 export PATH=$PATH:$HOME/iRODS/clients/icommands/bin

 iinit #this will prompt you for a bunch of information

Enter the host name (DNS) of the server to connect to:  mozart.arl.arizona.edu

Enter the port number:  1247

Enter your irods user name:  <your login name from UAGC/BCF>

Enter your irods zone:  BCFZone

Enter your current iRODS password:  <your password from UAGC/BCF>

 

#now your session should start

ils #lists all of the folders in your iRODS account

icd 14140709_SN885_0197_Lane1SideA_Project_Davalos_RNA #change directory

iget -r 140709_SN885_0197_Lane1SideA_Project_Davalos_RNA /Volumes/Spare/ 

#iget -r copies the folder you want to whatever directory you want

Okay now all of the data should be on your computer.

Now I need to install Trinity:

#install homebrew if you do not already have it

ruby -e "$(curl -fsSL https://raw.github.com/Homebrew/homebrew/go/install)"

#now install programs necessary for trinity to run on your mac

brew install homebrew/science/bowtie

brew install git

#to allow for new installation of compiler

brew tap homebrew/dupes

brew install apple-gcc42

#okay guess I can't do that because it requires an operating system higher than Mac OSX Lion...

Yes, you read that correctly. Our operating system is still pre-lion. Seems like I will have to wait until our operating system is figured out.

Once we figure this out though, remember this link: http://sourceforge.net/p/trinityrnaseq/mailman/message/31892980/

It allows for trinity to be installed on Mac (even though it is compiled for Linux).