# Data Management – SRA Submission Oly GBS Batch Submission

An earlier attempt at submitting these files failed.

I re-uploaded the failed files (indicated in my previous notebook entry linked above) and tried again.

It failed again, despite having successfully uploaded just minutes before.

I re-uploaded that “missing” file and tried again.

This time, it succeeded (and no end-of-stream error for the 1SN_1A file!)!

Will post here with the SRA accession number once it goes live!

# Computing – Owl Partially Restored

Heard back from Synology and they indicated I should click the “Repair” option to fix the System Partition Failed error message seen previously.

I did that and our data is now accessible again. However, all the user account info, scheduled tasks (e.g. Glacier backups, notebook backup script), IP configurations, mail configurations, etc. have all been reset.

I downloaded/installed the various packages needed to have the server accessible via the web and configured the IP address settings.

Have a note out to Synology to see if the configurations can be restored somehow. Once I hear back, we’ll get user accounts re-established.

Below is a chronological set of screen caps of the repair process:

Our data is still here! This is before performing the “Repair” operation, btw. It seems it just required some time to re-populate directory structure.

Still getting a “degraded” error message, but all drives appear normal. However, Disk 3 in the DX513 is not showing; possible cause for “degraded” status?

Set up manual IP settings by expanding the “LAN 1″ connection.

# Data Management – SRA Submission Oly GBS Batch Submission Fail

He noticed that the SRA no longer wants “raw data dumps” (i.e. the type of submission I made before). So, that means I had to prepare the demultiplexed files provided by BGI to actually submit to the SRA.

Last week, I uploaded all 192 of the files via FTP. It took over 10hrs.

(FTP tips: – Use ftp -i to initiate FTP. – Then use open ftp.address.IP to connect. – Then can use mput with regular expressions to upload multiple files)

Today, I created a batch BioSample submission:

Initiated the submission process (Ummm, this looks like it’s going to take awhile…):

Aaaaaand, it failed:

It seems like the FTP failed at some point, as there’s nothing about those seven files that would separate them from the remaining 185 files. Additional support for FTP failure is that the 1SN_1A_1.fq.gz error message makes it sound like only part of the file got transferred.

I’ll retrieve those files from our UW Google Drive (since their original home on Owl is still down) and get them trasnferred to the SRA.

# Troubleshooting – Synology NAS (Owl) Down After Update

TL;DR – Server didn’t recover after firmware update last night. “Repair” is an option listed in the web interface, but I want to confirm with Synology what will happen if/when I click that button…

The data on Owl is synced here (Google Drive): UW Google Drive

However, not all of Owl was fully synced at the time of this failure, so it seems like a decent amount of data is not accessible. Inaccessible data is mostly from individual user directories.

All high-throughput sequencing is also backed up to Amazon Glacier, so we do have all of that data.

Here is what happened, in chronological order:

1. Updated DSM via web interface in “Update & Restore”. Did NOT perform manual install.
2. System became inaccessible via web interface and Synology Assistant.
3. The physical unit showed blue, flashing power light and green flashing LAN1 light.
4. No other lights were illuminated (this includes no lights for any of the drive bays).
5. The attached expansion unit (DX513) showed steady blue power light, steady green lights on all drive bays, and steady green eSATA light.
6. I powered down both units via the DS1812+ power button.
7. I turned on the both units via the DS1812+ power button.
8. Both units returned to their previous status and were still inaccessible via the web interface and Synology Assistant.
9. I powered down both units via the DS1812+ power button.
10. I removed all drives from both units.
11. I turned on the both units via the DS1812+ power button.
12. I connected to the DS1812+ via Synology Assistant. A message indicated “No Hard Disk Found on 1812+”.
13. I powered down both units via the DS1812+ power button.
14. I added a single HDD to the DS1812+.
15. I turned on the both units via the DS1812+ power button.
16. I connected to the DS1812+ via Synology Assistant. I was prompted to install the latest DSM. I followed the steps and created a new admin account. Now the system shows 7 drives in the DS1812+ with a message: “System Partition Failed; Healthy”. Disk 1 shows a “Normal” status; this is the disk that I used to re-install DSM in Step 14. Additionally, the system shows one unused disk in the DX513.
17. I powered down both units via the web interface.
18. I removed Disk 1 from DS1812+.
19. I turned on the both units via the DS1812+ power button.
20. The DS1812+ returns to its initial state as described in Step 3.
21. I powered down both units via the DS1812+ power button.
22. I returned Disk 1 to its bay.
23. I turned on the both units via the DS1812+ power button.
24. There’s an option to “Repair” the Volume, but I’m not comfortable doing so until I discuss the in/outs of this with Synology. Submitted a tech support ticket with Synology.

Below are pictures of the entire process, for reference.

Server status when I arrived to lab this morning.

Pulled the HDDs from both units, in an attempt to be able to connect via Synology Assistant.

Units w/o HDDs.

No HDDs in units made the server detectable via Synology Assistant, but it indicates “Not installed” in the “Status” column…

Successfully connected, but the DS1812+ indicates no HDDs installed.

Added a single HDD back to the DS1812+. Notice, the drive light is green and the “Status” light is amber. This is actually an improvement over what I saw when I arrived.

Added back a single HDD to the DS1812+ and now have this setup menu.

I’m prompted to install the Synology DSM.

Installing DSM. This “Formatting system partition” message might be related to the eventual error message that I see (“System Partition Failed”) after this is all set up…

Prompted to create an admin account. This does not bode well, since this is behaving like a brand new installation (i.e. no record of the previous configuration, users, etc.).

Continuing set up.

All set up…

Added all the HDDs back and detected via Synology Assistant.

This shows that there are no other users – i.e. previous configuration is not detected.

After putting all the HDDs back in, got this message after logging in.

Looking at the Storage info in DSM; seems bad.

Physically, the drives all look fine (green lights on all drive bays), despite the indication in the DSM about “System Partition Failed” for all of them (except Disk 1). The expansion unit’s bay lights are actually all green, but were actively being read at the time of picture (i.e. flashing) so the image didn’t capture all of them being green. Amber light on expansion unit reflects what was seen in the DSM – the middle drive is “Not initialized”. Note, the drive is actually inserted, but the handle has been released.

This is how I left the system. Notice that after rebooting, the expansion unit no longer shows that “Not initialized” message for Disk 3. Instead, Disk 3 is now detected as installed, but not used…

# Computing – Oly BGI GBS Reproducibility; fail?

OK, so things have improved since the last attempt at getting this BGI script to run and demultiplex the raw data.

I played around with the index.lst file format (based on the error I received last time, it seemed like a good possibility that the file formatting was incorrect) and actually got the script to run to completion! Granted, it took over 16hrs (!!), but it completed!

See the Jupyter notebook link below.

Results:

Well, although the script finished and kicked out all the demultiplexed FASTQ files, the contents of the FASTQ files don’t match (the read counts differ between these results and the BGI files) the original set of demultiplexed files. I’m not entirely sure if this is to be expected or not, since the script allows for a single nucleotide mismatch when demultiplexing. Is it possible that the mismatch could be interpreted slightly differently each time this is run? I’m not certain.

Theoretically, you should get the same results every time…

Maybe I’ll re-run this again over the weekend and see how the results compare to this run and the original BGI demultiplexing…

Jupyter notebook (GitHub): 20170314_docker_Oly_BGI_GBS_demultiplexing_reproducibility.ipynb

Jupyter notebook (may be easier to view in GitHub link above):

# Computing – Oly BGI GBS Reproducibility Fail (but, less so than last time)…

Well, my previous attempt at reproducing the demultiplexing that BGI performed was an exercise in futility. BGI got back to me with the following message:

Hi Sam,

We downloaded it and it seems fine when compiling. You can compile it with the below command under Linux system.

tar -zxvf ReSeqTools_XXX.tar.gz ; cd iTools_Code; chmod 775 iTools ; ./ iTools -h

I gave that whirl and got the following message:

Error opening terminal: xterm

Some internet searching got me sucked into a useless black hole about 64 bit systems running 32 bit programs and enabling the 64 bit kernel on Mac OS X 10.7.5 (Lion) since it’s not enabled by default and on and on. In the end, I can’t seem to enable the 64 bit kernel on my Mac Pro, likely due to hardware limitations related to the graphics card and/or displays that are connected.

Anyway, I decided to try getting this program installed again, using a Docker container (instead of trying to install locally on my Mac).

Results:

It didn’t work again, but for a different reason! Despite the instructions in the readme file provided with iTools, you don’t actually need to run make! All that has to be done is unzipping the tarball!! However, despite figuring this out, the program fails with the following error message: “Warming : sample double in this INDEX Files. Sample ID: OYSzenG1AAD96FAAPEI-109; please renamed it diff” (note: this is copied/pasted – the spelling errors are note mine). So, I think there’s something wrong with the formatting of the index file that BGI provided me with.

See the Jupyter notebook linked below to see what I tried.

Jupyter notebook (GitHub): 20170314_docker_Oly_BGI_GBS_demultiplexing_reproducibility.ipynb

# Computing – Oly BGI GBS Reproducibility Fail

Since we’re preparing a manuscript that relies on BGI’s manipulation/handling of the genotype-by-sequencing data, I attempted to could reproduce the demultiplexing steps that BGI used in order to perform the SNP/genotyping on these samples.

The key word in the above sentence is “attempted.” Ugh, what a massive waste of time it turned out to be. I’ve contacted BGI to get some help on this.

In the meantime, here’s a brief (actually, not as brief as I’d like) rundown of my struggles.

The demultiplexing software that BGI used is something called “iTools” which is bundled in this GitHub repo: Resqtools

To demutliplex, they ran a script called: split.sh

The script seems fairly straightforward. Here is what it contains:

iTools Fqtools splitpool \
-Index index.lst \
-Flag enzyme.txt \
-MisMatch \
-OutDir split


It tells the iTools program to use the Fqtools tool “splitpool” to operate on a pair of gzipped FASTQ files. It also utilizes an index file (index.lst) which contains all the barcodes needed to identify, and separate, the individual samples that were combined prior to sequencing.

The first bump in the road is the -Flag enzyme.txt portion of the code. BGI did not provide me with this file. I recently requested them to send me it (or its contents, since I suspected it was only a single line text file). They sent me the contents of the file:

CAGC
CTGC

The next problem is neither of those two sequences are the recognition site for the enzyme that was (supposedly) used: ApeKI. The recognition site for ApeKI is: GCWGC

Regardless, I decided to see if I could reproduce the demultiplexing using the info they’d provided me.

I cloned the Resqtools repo, changed into the Reseqtools/iTools directory and typed make.

This resulted in an error informing me that it could not find boost/spirit/core.hpp

Tried to run make in the Reseqtools/iTools directory and got the same error. Realized iTools might not be searching the system \$PATH (this turned out to be correct), so I moved the contents of the Boost folder to the iTools, ran make and got the same error. Turns out, the newest version of Boost doesn’t have that core.hpp file any more. Looking at the iTools documentation, iTools was built around Boost 1.44. OMG…

Downloaded Boost 1.44 and went through the same steps as above. This eliminated the missing core.hpp error!

But, of course, led to another error. The error:

"Threading support unavaliable: it has been explicitly disabled with BOOST_DISABLE_THREADS"

That was related to something with newer versions of the GCC compiler (this is, essentially, built into the computer; it’s not worth trying to install/use old versions of GCC) trying to work with old versions of Boost. Found a patch for a config file here: libstdcpp3.hpp.patch

I made the appropriate edits to the file as shown in that link and ran make and it almost worked!

The current error is:

./src/Variants/soapsv-v1.02/include.h:15:16: fatal error: gd.h: No such file or directory

I gave up and contacted BGI to see if they can get me a functional version of iTools…

# FASTQC – Oly BGI GBS Raw Illumina Data Demultiplexed

Last week, I ran the two raw FASTQ files through FastQC. As expected, FastQC detected “errors”. These errors are due to the presence of adapter sequences, barcodes, and the use of a restriction enzyme (ApeKI) in library preparation. In summary, it’s not surprising that FastQC was not please with the data because it’s expecting a “standard” library prep that’s already been trimmed and demultiplexed.

However, just for comparison, I ran the demultiplexed files through FastQC. The Jupyter notebook is linked (GitHub) and embedded below. I recommend viewing the Jupyter notebook on GitHub for easier viewing.

Results:

Pretty much the same, but with slight improvements due to removal of adapter and barcode sequences. The restriction site still leads to FastQC to report errors, which is expected.

Links to all of the FastQC output files are linked at the bottom of the notebook.

Jupyter notebook (GitHub): 20170306_docker_fastqc_demultiplexed_bgi_oly_gbs.ipynb

# FASTQC – Oly BGI GBS Raw Illumina Data

In getting things prepared for the manuscript we’re writing about the Olympia oyster genotype-by-sequencing data from BGI, I felt we needed to provide a FastQC analysis of the raw data (since these two files are what we submitted to the NCBI short read archive) to provide support for the Technical Validation section of the manuscript.

Below, is the Jupyter notebook I used to run the FastQC analysis on the two files. I’ve embedded for quick viewing, but it might be easier to view the notebook via the GitHub link.

Results:

Well, I realized that running FastQC on the raw data might not reveal anything all too helpful. The reason for this is that the adaptor and barcode sequences are still present on all the reads. This will lead to over-representation of these sequences in all of the samples, which, in turn, will skew FastQC’s intepretation of the read qualities. For comparison, I’ll run FastQC on the demultiplexed data provided by BGI and see what the FastQC report looks like on trimmed data.

However, I’ll need to discuss with Steven about whether or not providing the FastQC analysis is worthwhile as part of the “technical validation” aspect of the manuscript. I guess it can’t hurt to provide it, but I’m not entirely sure that the FastQC report provides any real information regarding the quality of the sequencing reads that we received…

Jupyter notebook (GitHub): 20170301_docker_fastqc_nondemultiplexed_bgi_oly_gbs.ipynb

# Goals – March 2017

Goal, singular: Get Oly GBS manuscript completed/submitted.

Oh, actually, there is another, smaller goal that will be very difficult to achieve: win Pub-a-Thon. Jay’s taken a massive lead and has a nearly complete manuscript ready for submission. His manuscript is pretty well fleshed out, so it’ll be very difficult to surpass  him at this point. However, I’m always up for a challenge, so I’ll see what I can do…

Anyway, back to my main goal of completing my manuscript.

This should be do-able. I’ve completed the SRA submission process for the raw sequencing data. The stuff that remains is as follows:

• Generate FASTQC analysis on FASTQ files (this is currently running – takes awhile)
• Try to replicate BGI’s FASTQ demultiplexing pipeline to verify that it is functional
• Make decisions with Steven (and Brent?) about what information tables should contain
• Write

The beauty of submitting this to the journal Scientific Data, is that it doesn’t require in-depth analysis of your data sets. It merely requires an examination of the data to ensure its integrity, as well as a cursory assessment of the data to evaluate it’s usefulness to the scientific community. No need to delve deeper into the data and attempt to interpret, or draw conclusions about, what the data might mean; that can be left to other researchers who deem this data worthwhile to explore.