Tag Archives: NGS sequencing

Data Received – Olympia oyster PacBio Data

Back in December 2016, we sent off Ostrea lurida DNA to the UW PacBio sequencing facility. This is an attempt to fill in the gaps left from the BGI genome sequencing project.

See the GitHub Wiki dedicated to this for an overview of this UW PacBio sequencing.

I downloaded the data to http://owl.fish.washington.edu/nightingales/O_lurida/20170323_pacbio/ using the required browser plugin, Aspera Connect. Technically, saving the data to a subfolder within a given species’ data folder goes against our data management plan (DMP) for high-throughput sequencing data, but the sequencing data output is far different than what we normally receive from an Illumina sequencing run. Instead of a just FASTQ files, we received the following from each PacBio SMRT cell we had run (we had 10 SMRT cells run):

├── Analysis_Results
│   ├── m170211_224036_42134_c101073082550000001823236402101737_s1_X0.1.bax.h5
│   ├── m170211_224036_42134_c101073082550000001823236402101737_s1_X0.2.bax.h5
│   ├── m170211_224036_42134_c101073082550000001823236402101737_s1_X0.3.bax.h5
│   └── m170211_224036_42134_c101073082550000001823236402101737_s1_X0.bas.h5
├── filter
│   ├── data
│   │   ├── control_reads.cmp.h5
│   │   ├── control_results_by_movie.csv
│   │   ├── data.items.json
│   │   ├── data.items.pickle
│   │   ├── filtered_regions
│   │   │   ├── m170211_224036_42134_c101073082550000001823236402101737_s1_X0.1.rgn.h5
│   │   │   ├── m170211_224036_42134_c101073082550000001823236402101737_s1_X0.2.rgn.h5
│   │   │   └── m170211_224036_42134_c101073082550000001823236402101737_s1_X0.3.rgn.h5
│   │   ├── filtered_regions.fofn
│   │   ├── filtered_subread_summary.csv
│   │   ├── filtered_subreads.fasta
│   │   ├── filtered_subreads.fastq
│   │   ├── filtered_summary.csv
│   │   ├── nocontrol_filtered_subreads.fasta
│   │   ├── post_control_regions.chunk001of003
│   │   │   └── m170211_224036_42134_c101073082550000001823236402101737_s1_X0.1.rgn.h5
│   │   ├── post_control_regions.chunk002of003
│   │   │   └── m170211_224036_42134_c101073082550000001823236402101737_s1_X0.3.rgn.h5
│   │   ├── post_control_regions.chunk003of003
│   │   │   └── m170211_224036_42134_c101073082550000001823236402101737_s1_X0.2.rgn.h5
│   │   ├── post_control_regions.fofn
│   │   └── slots.pickle
│   ├── index.html
│   ├── input.fofn
│   ├── input.xml
│   ├── log
│   │   ├── P_Control
│   │   │   ├── align.cmpH5.Gather.log
│   │   │   ├── align.plsFofn.Scatter.log
│   │   │   ├── align_001of003.log
│   │   │   ├── align_002of003.log
│   │   │   ├── align_003of003.log
│   │   │   ├── noControlSubreads.log
│   │   │   ├── summaryCSV.log
│   │   │   ├── updateRgn.noCtrlFofn.Gather.log
│   │   │   ├── updateRgn_001of003.log
│   │   │   ├── updateRgn_002of003.log
│   │   │   └── updateRgn_003of003.log
│   │   ├── P_ControlReports
│   │   │   └── statsJsonReport.log
│   │   ├── P_Fetch
│   │   │   ├── adapterRpt.log
│   │   │   ├── overviewRpt.log
│   │   │   └── toFofn.log
│   │   ├── P_Filter
│   │   │   ├── filter.rgnFofn.Gather.log
│   │   │   ├── filter.summary.Gather.log
│   │   │   ├── filter_001of003.log
│   │   │   ├── filter_002of003.log
│   │   │   ├── filter_003of003.log
│   │   │   ├── subreadSummary.log
│   │   │   ├── subreads.subreadFastq.Gather.log
│   │   │   ├── subreads.subreads.Gather.log
│   │   │   ├── subreads_001of003.log
│   │   │   ├── subreads_002of003.log
│   │   │   └── subreads_003of003.log
│   │   ├── P_FilterReports
│   │   │   ├── loadingRpt.log
│   │   │   ├── statsRpt.log
│   │   │   └── subreadRpt.log
│   │   ├── master.log
│   │   └── smrtpipe.log
│   ├── metadata.rdf
│   ├── results
│   │   ├── adapter_observed_insert_length_distribution.png
│   │   ├── adapter_observed_insert_length_distribution_thumb.png
│   │   ├── control_non-control_readlength.png
│   │   ├── control_non-control_readlength_thumb.png
│   │   ├── control_non-control_readquality.png
│   │   ├── control_non-control_readquality_thumb.png
│   │   ├── control_report.html
│   │   ├── control_report.json
│   │   ├── filter_reports_adapters.html
│   │   ├── filter_reports_adapters.json
│   │   ├── filter_reports_filter_stats.html
│   │   ├── filter_reports_filter_stats.json
│   │   ├── filter_reports_filter_subread_stats.html
│   │   ├── filter_reports_filter_subread_stats.json
│   │   ├── filter_reports_loading.html
│   │   ├── filter_reports_loading.json
│   │   ├── filtered_subread_report.png
│   │   ├── filtered_subread_report_thmb.png
│   │   ├── overview.html
│   │   ├── overview.json
│   │   ├── post_filter_readlength_histogram.png
│   │   ├── post_filter_readlength_histogram_thumb.png
│   │   ├── post_filterread_score_histogram.png
│   │   ├── post_filterread_score_histogram_thumb.png
│   │   ├── pre_filter_readlength_histogram.png
│   │   ├── pre_filter_readlength_histogram_thumb.png
│   │   ├── pre_filterread_score_histogram.png
│   │   └── pre_filterread_score_histogram_thumb.png
│   ├── toc.xml
│   └── workflow
│       ├── P_Control
│       │   ├── align.cmpH5.Gather.sh
│       │   ├── align.plsFofn.Scatter.sh
│       │   ├── align_001of003.sh
│       │   ├── align_002of003.sh
│       │   ├── align_003of003.sh
│       │   ├── noControlSubreads.sh
│       │   ├── summaryCSV.sh
│       │   ├── updateRgn.noCtrlFofn.Gather.sh
│       │   ├── updateRgn_001of003.sh
│       │   ├── updateRgn_002of003.sh
│       │   └── updateRgn_003of003.sh
│       ├── P_ControlReports
│       │   └── statsJsonReport.sh
│       ├── P_Fetch
│       │   ├── adapterRpt.sh
│       │   ├── overviewRpt.sh
│       │   └── toFofn.sh
│       ├── P_Filter
│       │   ├── filter.rgnFofn.Gather.sh
│       │   ├── filter.summary.Gather.sh
│       │   ├── filter_001of003.sh
│       │   ├── filter_002of003.sh
│       │   ├── filter_003of003.sh
│       │   ├── subreadSummary.sh
│       │   ├── subreads.subreadFastq.Gather.sh
│       │   ├── subreads.subreads.Gather.sh
│       │   ├── subreads_001of003.sh
│       │   ├── subreads_002of003.sh
│       │   └── subreads_003of003.sh
│       ├── P_FilterReports
│       │   ├── loadingRpt.sh
│       │   ├── statsRpt.sh
│       │   └── subreadRpt.sh
│       ├── Workflow.details.dot
│       ├── Workflow.details.html
│       ├── Workflow.details.svg
│       ├── Workflow.profile.html
│       ├── Workflow.rdf
│       ├── Workflow.summary.dot
│       ├── Workflow.summary.html
│       └── Workflow.summary.svg
├── filtered_subreads.fasta.gz
├── filtered_subreads.fastq.gz
├── m170211_224036_42134_c101073082550000001823236402101737_s1_X0.metadata.xml
└── nocontrol_filtered_subreads.fasta.gz

That’s 20 directories and 127 files – for a single SMRT cell!

Granted, there is the familiar FASTQ file (filtered_subreads.fastq), which is what will likely be used for downstream analysis, but it’s hard to make a decision on how we manage this data under the guidelines of our current DMP. It’s possible we might separate data files from the numerous other files (the other files are, essentially, metadata), but we need to decide which file type(s) (e.g. .h5 files, .fastq files) will server as the data files people will rely on for analysis. So, for the time being, this will be how the data is stored.

I’ll update the readme file to reflect the addition of the top level folders (e.g. ../20170323_pacbio/170210_PCB-CC_MS_EEE_20kb_P6v2_D01_1/).

I’ll also update the GitHub Wiki

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Bioinformatics – Trimmomatic/FASTQC on C.gigas Larvae OA NGS Data

Previously trimmed the first 39 bases of sequence from reads from the BS-Seq data in an attempt to improve our ability to map the reads back to the C.gigas genome. However, Mac (and Steven) noticed that the last ~10 bases of all the reads showed a steady increase in the %G, suggesting some sort of bias (maybe adaptor??):

Although I didn’t mention this previously, the figure above also shows an odd “waves” pattern that repeats in all bases except for G. Not sure what to think of that…

Quick summary of actions taken (specifics are available in Jupyter notebook below):

  • Trim first 39 bases from all reads in all raw sequencing files.
  • Trim last 10 bases from all reads in raw sequencing files
  • Concatenate the two sets of reads (400ppm and 1000ppm treatments) into single FASTQ files for Steven to work with.

Raw sequencing files:

Notebook Viewer: 20150521_Cgigas_larvae_OA_Trimmomatic_FASTQC

Jupyter (IPython) notebook: 20150521_Cgigas_larvae_OA_Trimmomatic_FASTQC.ipynb

 

 

Output files

Trimmed, concatenated FASTQ files
20150521_trimmed_2212_lane2_400ppm_GCCAAT.fastq.gz
20150521_trimmed_2212_lane2_1000ppm_CTTGTA.fastq.gz

 

FASTQC files
20150521_trimmed_2212_lane2_400ppm_GCCAAT_fastqc.html
20150521_trimmed_2212_lane2_400ppm_GCCAAT_fastqc.zip

20150521_trimmed_2212_lane2_1000ppm_CTTGTA_fastqc.html
20150521_trimmed_2212_lane2_1000ppm_CTTGTA_fastqc.zip

 

Example of FASTQC analysis pre-trim:

 

 

Example FASTQC post-trim (from 400ppm data):

 

Trimming has removed the intended bad stuff (inconsistent sequence in the first 39 bases and rise in %G in the last 10 bases). Sequences are ready for further analysis for Steven.

However, we still see the “waves” pattern with the T, A and C. Additionally, we still don’t know what caused the weird inconsistencies, nor what sequence is contained therein that might be leading to that. Will contact the sequencing facility to see if they have any insight.

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Quality Trimming – LSU C.virginica Oil Spill MBD BS-Seq Data

Jupyter (IPython) Notebook: 20150414_C_virginica_LSU_Oil_Spill_Trimmomatic_FASTQC.ipynb

NBviewer: 20150414_C_virginica_LSU_Oil_Spill_Trimmomatic_FASTQC.ipynb

Trimmed FASTQC

NB3 No oil Index – ACAGTG

20150414_trimmed_2112_lane1_ACAGTG_L001_R1_001_fastqc.html
20150414_trimmed_2112_lane1_ACAGTG_L001_R1_002_fastqc.html

NB6 No oil Index – GCCAAT

20150414_trimmed_2112_lane1_GCCAAT_L001_R1_001_fastqc.html
20150414_trimmed_2112_lane1_GCCAAT_L001_R1_002_fastqc.html

NB11 No oil Index – CAGATC

20150414_trimmed_2112_lane1_CAGATC_L001_R1_001_fastqc.html
20150414_trimmed_2112_lane1_CAGATC_L001_R1_002_fastqc.html
20150414_trimmed_2112_lane1_CAGATC_L001_R1_003_fastqc.html

HB2 25,000ppm oil Index – ATCACG

20150414_trimmed_2112_lane1_ATCACG_L001_R1_001_fastqc.html
20150414_trimmed_2112_lane1_ATCACG_L001_R1_002_fastqc.html
20150414_trimmed_2112_lane1_ATCACG_L001_R1_003_fastqc.html

HB16 25,000ppm oil Index – TTAGGC

20150414_trimmed_2112_lane1_TTAGGC_L001_R1_001_fastqc.html
20150414_trimmed_2112_lane1_TTAGGC_L001_R1_002_fastqc.html

HB30 25,000ppm oil Index – TGACCA

20150414_trimmed_2112_lane1_TGACCA_L001_R1_001_fastqc.html

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Sequence Data Analysis – LSU C.virginica Oil Spill MBD BS-Seq Data

Performed some rudimentary data analysis on the new, demultiplexed data downloaded earlier today:

2112_lane1_ACAGTG_L001_R1_001.fastq.gz
2112_lane1_ACAGTG_L001_R1_002.fastq.gz
2112_lane1_ATCACG_L001_R1_001.fastq.gz
2112_lane1_ATCACG_L001_R1_002.fastq.gz
2112_lane1_ATCACG_L001_R1_003.fastq.gz
2112_lane1_CAGATC_L001_R1_001.fastq.gz
2112_lane1_CAGATC_L001_R1_002.fastq.gz
2112_lane1_CAGATC_L001_R1_003.fastq.gz
2112_lane1_GCCAAT_L001_R1_001.fastq.gz
2112_lane1_GCCAAT_L001_R1_002.fastq.gz
2112_lane1_TGACCA_L001_R1_001.fastq.gz
2112_lane1_TTAGGC_L001_R1_001.fastq.gz
2112_lane1_TTAGGC_L001_R1_002.fastq.gz

 

Compared total amount of data (in gigabytes) generated from each index. The commands below send the output of the ‘ls -l’ command to awk. Awk sums the file sizes, found in the 5th field ($5) of the ‘ls -l’ command, then prints the sum, divided by 1024^3 to convert from bytes to gigabytes.

Index: ACAGTG

$ls -l 2112_lane1_AC* | awk '{sum += $5} END {print sum/1024/1024/1024}'
1.49652

 

Index: ATCACG

$ls -l 2112_lane1_AT* | awk '{sum += $5} END {print sum/1024/1024/1024}'
3.02269

 

Index: CAGATC

$ls -l 2112_lane1_CA* | awk '{sum += $5} END {print sum/1024/1024/1024}'
3.49797

 

Index: GCCAAT

$ls -l 2112_lane1_GC* | awk '{sum += $5} END {print sum/1024/1024/1024}'
2.21379

 

Index: TGACCA

$ls -l 2112_lane1_TG* | awk '{sum += $5} END {print sum/1024/1024/1024}'
0.687374

 

Index: TTAGGC

$ls -l 2112_lane1_TT* | awk '{sum += $5} END {print sum/1024/1024/1024}'
2.28902

 

Ran FASTQC on the following files downloaded earlier today. The FASTQC command is below. This command runs FASTQC in a for loop over any files that begin with “2212_lane2_C” or “2212_lane2_G” and outputs the analyses to the Arabidopsis folder on Eagle:

$for file in /Volumes/nightingales/C_virginica/2112_lane1_[ATCG]*; do fastqc "$file" --outdir=/Volumes/Eagle/Arabidopsis/; done

 

From within the Eagle/Arabidopsis folder, I renamed the FASTQC output files to prepend today’s date:

$for file in 2112_lane1_[ATCG]*; do mv "$file" "20150413_$file"; done

 

Then, I unzipped the .zip files generated by FASTQC in order to have access to the images, to eliminate the need for screen shots for display in this notebook entry:

$for file in 20150413_2112_lane1_[ATCG]*.zip; do unzip "$file"; done

 

The unzip output retained the old naming scheme, so I renamed the unzipped folders:

$for file in 2112_lane1_[ATCG]*; do mv "$file" "20150413_$file"; done

 

The FASTQC results are linked below:

20150413_2112_lane1_ACAGTG_L001_R1_001_fastqc.html
20150413_2112_lane1_ACAGTG_L001_R1_002_fastqc.html
20150413_2112_lane1_ATCACG_L001_R1_001_fastqc.html
20150413_2112_lane1_ATCACG_L001_R1_002_fastqc.html
20150413_2112_lane1_ATCACG_L001_R1_003_fastqc.html
20150413_2112_lane1_CAGATC_L001_R1_001_fastqc.html
20150413_2112_lane1_CAGATC_L001_R1_002_fastqc.html
20150413_2112_lane1_CAGATC_L001_R1_003_fastqc.html
20150413_2112_lane1_GCCAAT_L001_R1_001_fastqc.html
20150413_2112_lane1_GCCAAT_L001_R1_002_fastqc.html
20150413_2112_lane1_TGACCA_L001_R1_001_fastqc.html
20150413_2112_lane1_TTAGGC_L001_R1_001_fastqc.html
20150413_2112_lane1_TTAGGC_L001_R1_002_fastqc.html

 

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Sequence Data Analysis – C.gigas Larvae OA BS-Seq Data

Compared total amount of data generated from each index. The commands below send the output of the ‘ls -l’ command to awk. Awk sums the file sizes, found in the 5th field ($5) of the ‘ls -l’ command, then prints the sum, divided by 1024^3 to convert from bytes to gigabytes.

Index: CTTGTA

$ ls -l 2212_lane2_[C]* | awk '{sum += $5} END {print sum/1024/1024/1024}'
5.33341

Index: GCCAAT
$ ls -l 2212_lane2_[G]* | awk '{sum += $5} END {print sum/1024/1024/1024}'
7.00596

There’s ~1.4x data in the GCCAAT files.

 

Ran FASTQC on the following files downloaded earlier today:

2212_lane2_CTTGTA_L002_R1_001.fastq.gz
2212_lane2_CTTGTA_L002_R1_002.fastq.gz
2212_lane2_CTTGTA_L002_R1_003.fastq.gz
2212_lane2_CTTGTA_L002_R1_004.fastq.gz
2212_lane2_GCCAAT_L002_R1_001.fastq.gz
2212_lane2_GCCAAT_L002_R1_002.fastq.gz
2212_lane2_GCCAAT_L002_R1_003.fastq.gz
2212_lane2_GCCAAT_L002_R1_004.fastq.gz
2212_lane2_GCCAAT_L002_R1_005.fastq.gz
2212_lane2_GCCAAT_L002_R1_006.fastq.gz

 

The FASTQC command is below. This command runs FASTQC in a for loop over any files that begin with “2212_lane2_C” or “2212_lane2_G” and outputs the analyses to the Arabidopsis folder on Eagle:

$for file in /Volumes/nightingales/C_gigas/2212_lane2_[CG]*; do fastqc "$file" --outdir=/Volumes/Eagle/Arabidopsis/; done

 

From within the Eagle/Arabidopsis folder, I renamed the FASTQC output files to prepend today’s date:

$for file in 2212_lane2_[GC]*; do mv "$file" "20150413_$file"; done

 

Then, I unzipped the .zip files generated by FASTQC in order to have access to the images, to eliminate the need for screen shots for display in this notebook entry:

$for file in 20150413_2212_lane2_[CG]*.zip; do unzip "$file"; done

 

The unzip output retained the old naming scheme, so I renamed the unzipped folders:

$for file in 2212_lane2_[GC]*; do mv “$file” “20150413_$file”; done

 

The FASTQC results are linked below:

20150413_2212_lane2_CTTGTA_L002_R1_001_fastqc.html

20150413_2212_lane2_CTTGTA_L002_R1_002_fastqc.html
20150413_2212_lane2_CTTGTA_L002_R1_003_fastqc.html
20150413_2212_lane2_CTTGTA_L002_R1_004_fastqc.html
20150413_2212_lane2_GCCAAT_L002_R1_001_fastqc.html
20150413_2212_lane2_GCCAAT_L002_R1_002_fastqc.html
20150413_2212_lane2_GCCAAT_L002_R1_003_fastqc.html
20150413_2212_lane2_GCCAAT_L002_R1_004_fastqc.html
20150413_2212_lane2_GCCAAT_L002_R1_005_fastqc.html
20150413_2212_lane2_GCCAAT_L002_R1_006_fastqc.html

 

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Sequence Data – C.gigas OA Larvae BS-Seq Demultiplexed

I had previously contacted Doug Turnbull at the Univ. of Oregon Genomics Core Facility for help demultiplexing this data, as it was initially returned to us as a single data set with “no index” (i.e. barcode) set for any of the libraries that were sequenced. As it turns out, when multiplexed libraries are sequenced using the Illumina platform, an index read step needs to be “enabled” on the machine for sequencing. Otherwise, the machine does not perform the index read step (since it wouldn’t be necessary for a single library). Surprisingly, the sample submission form for the Univ. of Oregon Genomics Core Facility  doesn’t request any information regarding whether or not a submitted sample has been multiplexed. However, by default, they enable the index read step on all sequencing runs. I provided them with the barcodes and they demultiplexed them after the fact.

I downloaded the new, demultiplexed files to Owl/nightingales/C_gigas:

lane2_CTTGTA_L002_R1_001.fastq.gz
lane2_CTTGTA_L002_R1_002.fastq.gz
lane2_CTTGTA_L002_R1_003.fastq.gz
lane2_CTTGTA_L002_R1_004.fastq.gz
lane2_GCCAAT_L002_R1_001.fastq.gz
lane2_GCCAAT_L002_R1_002.fastq.gz
lane2_GCCAAT_L002_R1_003.fastq.gz
lane2_GCCAAT_L002_R1_004.fastq.gz
lane2_GCCAAT_L002_R1_005.fastq.gz
lane2_GCCAAT_L002_R1_006.fastq.gz

Notice that the file names now contain the corresponding index!

Renamed the files, to append the order number to the beginning of the file names:

$for file in lane2*; do mv "$file" "2212_$file"; done

New file names:

2212_lane2_CTTGTA_L002_R1_001.fastq.gz
2212_lane2_CTTGTA_L002_R1_002.fastq.gz
2212_lane2_CTTGTA_L002_R1_003.fastq.gz
2212_lane2_CTTGTA_L002_R1_004.fastq.gz
2212_lane2_GCCAAT_L002_R1_001.fastq.gz
2212_lane2_GCCAAT_L002_R1_002.fastq.gz
2212_lane2_GCCAAT_L002_R1_003.fastq.gz
2212_lane2_GCCAAT_L002_R1_004.fastq.gz
2212_lane2_GCCAAT_L002_R1_005.fastq.gz
2212_lane2_GCCAAT_L002_R1_006.fastq.gz

Updated the checksums.md5 file to include the new files (the command is written to exclude the previously downloaded files that are named “2212_lane2_NoIndex_”; the [^N] regex excludes any files that have a capital ‘N’ at that position in the file name):

$for file in 2212_lane2_[^N]*; do md5 "$file" >> checksums.md5; done

Updated the readme.md file to reflect the addition of these new files.

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Sequence Data – LSU C.virginica Oil Spill MBD BS-Seq Demultiplexed

I had previously contacted Doug Turnbull at the Univ. of Oregon Genomics Core Facility for help demultiplexing this data, as it was initially returned to us as a single data set with “no index” (i.e. barcode) set for any of the libraries that were sequenced. As it turns out, when multiplexed libraries are sequenced using the Illumina platform, an index read step needs to be “enabled” on the machine for sequencing. Otherwise, the machine does not perform the index read step (since it wouldn’t be necessary for a single library). Surprisingly, the sample submission form for the Univ. of Oregon Genomics Core Facility  doesn’t request any information regarding whether or not a submitted sample has been multiplexed. However, by default, they enable the index read step on all sequencing runs. I provided them with the barcodes and they demultiplexed them after the fact.

I downloaded the new, demultiplexed files to Owl/nightingales/C_virginica:

lane1_ACAGTG_L001_R1_001.fastq.gz
lane1_ACAGTG_L001_R1_002.fastq.gz
lane1_ATCACG_L001_R1_001.fastq.gz
lane1_ATCACG_L001_R1_002.fastq.gz
lane1_ATCACG_L001_R1_003.fastq.gz
lane1_CAGATC_L001_R1_001.fastq.gz
lane1_CAGATC_L001_R1_002.fastq.gz
lane1_CAGATC_L001_R1_003.fastq.gz
lane1_GCCAAT_L001_R1_001.fastq.gz
lane1_GCCAAT_L001_R1_002.fastq.gz
lane1_TGACCA_L001_R1_001.fastq.gz
lane1_TTAGGC_L001_R1_001.fastq.gz
lane1_TTAGGC_L001_R1_002.fastq.gz

Notice that the file names now contain the corresponding index!

Renamed the files, to append the order number to the beginning of the file names:

$for file in lane1*; do mv "$file" "2112_$file"; done

New file names:

2112_lane1_ACAGTG_L001_R1_001.fastq.gz
2112_lane1_ACAGTG_L001_R1_002.fastq.gz
2112_lane1_ATCACG_L001_R1_001.fastq.gz
2112_lane1_ATCACG_L001_R1_002.fastq.gz
2112_lane1_ATCACG_L001_R1_003.fastq.gz
2112_lane1_CAGATC_L001_R1_001.fastq.gz
2112_lane1_CAGATC_L001_R1_002.fastq.gz
2112_lane1_CAGATC_L001_R1_003.fastq.gz
2112_lane1_GCCAAT_L001_R1_001.fastq.gz
2112_lane1_GCCAAT_L001_R1_002.fastq.gz
2112_lane1_TGACCA_L001_R1_001.fastq.gz
2112_lane1_TTAGGC_L001_R1_001.fastq.gz
2112_lane1_TTAGGC_L001_R1_002.fastq.gz

Updated the checksums.md5 file to include the new files (the command is written to exclude the previously downloaded files that are named “2112_lane1_NoIndex_”; the [^N] regex excludes any files that have a capital ‘N’ at that position in the file name):

$for file in 2112_lane1_[^N]*; do md5 "$file" >> checksums.md5; done

Updated the readme.md file to reflect the addition of these new files.

 

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Bisulfite NGS Library – LSU C.virginica Oil Spill MBD Bisulfite DNA Sequencing Submission

Combined the following libraries in equal quantities (17ng each) to create a single, multiplexed sample for sequencing (LSU_Oil_01):

  • HB2 – 1 (ATCACG)
  • HB16 – 3 (TTAGGC)
  • HB30 – 4 (TGACCA)
  • NB3 – 5 (ACAGTG)
  • NB6 – 6 (GCCAAT)
  • NB11 – 7 (CAGATC)

Quantified pooled libraries using the Quant-iT dsDNA BR Kit (Invitrogen) with a FLx800 plate reader (BioTek). Used 1μL of the pooled sample, run in duplicate. Used 1uL of standards, run in duplicate.

Results:

pooled libraries = 6.575ng/μL

Will submit to University of Oregon Genomics Core Facility for 100bp, single end Illumina HiSeq2500 sequencing. They need 10nM of sample. For a library with average size range of 300-400bp, this requires a sample volume of 20uL with a concentration of 2.28ng/μL in a solution of 0.1% Tween20 in Buffer EB (Qiagen).

Combined 6.94μL of pooled libraries with 13.06 of 0.1% Tween20/EB solution.

Submitted sample LSU_Oil_01 to University of Oregon Genomics Core Facility via O/N FedEx on dry ice. Sample was assigned order # 2112.

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