From fcd5cb0be1b033a4c43934d1e2b6d4c4d4498829 Mon Sep 17 00:00:00 2001 From: Heli Juottonen Date: Thu, 10 Aug 2023 13:09:34 +0300 Subject: [PATCH] Add Ion Torrent exercises --- docs/IonTorrent/Exercises_day1.html | 1102 ++++++++++++++++ docs/IonTorrent/Exercises_day2.html | 1142 +++++++++++++++++ eLena_md/IonTorrent/.DS_Store | Bin 0 -> 6148 bytes .../IonTorrent/Exercises_IonTorrent_day1.Rmd | 219 ++++ .../IonTorrent/Exercises_IonTorrent_day2.Rmd | 222 ++++ .../Images/phenodata_IonTorrent.png | Bin 0 -> 62940 bytes .../IonTorrent/Images/taxbar_IonTorrent.png | Bin 0 -> 87439 bytes 7 files changed, 2685 insertions(+) create mode 100644 docs/IonTorrent/Exercises_day1.html create mode 100644 docs/IonTorrent/Exercises_day2.html create mode 100644 eLena_md/IonTorrent/.DS_Store create mode 100644 eLena_md/IonTorrent/Exercises_IonTorrent_day1.Rmd create mode 100644 eLena_md/IonTorrent/Exercises_IonTorrent_day2.Rmd create mode 100644 eLena_md/IonTorrent/Images/phenodata_IonTorrent.png create mode 100644 eLena_md/IonTorrent/Images/taxbar_IonTorrent.png diff --git a/docs/IonTorrent/Exercises_day1.html b/docs/IonTorrent/Exercises_day1.html new file mode 100644 index 0000000..9e912dc --- /dev/null +++ b/docs/IonTorrent/Exercises_day1.html @@ -0,0 +1,1102 @@ + + + + + + + + + + Exercises_day1.knit + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
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+

Day 1: Data pre-processing

+

This is an example workflow for analysing amplicon sequence data of +single-end reads from Ion Torrent sequencing.

+

The data consist of 16 samples of willow catkins to study +plant-associated bacteria. The samples are a subset from a larger study +that aimed to investigate the effect of pollinator visits on willow +catkin microbes (https://doi.org/10.1007/s00442-022-05285-7).

+

The samples here were collected from

+
    +
  • two sites +
      +
    • HP: no honeybee hives closeby
    • +
    • KEK: honeybee hives present at the site
    • +
  • +
  • two treatments (4 replicates each) +
      +
    • ps: willow catkins protected from pollinators by a mesh bag
    • +
    • c: control catkins that pollinators visited
    • +
  • +
+

Our aim is to find out if site and/or protection from pollinators +influenced the bacterial community in the willow catkins. All samples +were sequenced using Ion Torrent. The amplified region is the V6-V8 +region of the bacterial 16S rRNA gene and the length of the amplicon is +ca. 350 bp. The samples have been demultiplexed and barcodes removed, +and each FASTQ file corresponds to one sample.

+

Step 1. Start Chipster and open a session

+

Go to the Chipster +website and log in. In the session list, scroll down to Training +sessions and select +course_16S_rRNA_community_analysis_IonTorrent. This session has +16 zipped fastq files. Save your own copy of the session: click the +three dots in the Session info section on the lower right and +select Save a copy.

+

Step 2. Package all the fastq files in a tar +package

+

Select all the FASTQ files:

+
    +
  • Go to the List tab, click on the first file, keep the shift +key down and click on the last file.
  • +
  • Select the tool Utilities / Make a tar package.
  • +
  • Click Parameters, set +File name for tar package = zippedFastq, and close the +parameter window. Click Run and select Run Tool 1 +job.
  • +
+

Note that after this point in real life you can delete the individual +FASTQ files, because you can always open the tar package using the tool +Utilities / Extract .tar.gz file.

+

Step 3. Check the quality of reads with FastQC

+

Select the file zippedFastq.tar and the tool +Quality control / Read quality with MultiQC for many FASTQ files, +and click Run. Select the result file and click Open in new +tab.

+
How long are the reads (in the General Statistics section, click 
+Configure columns, select Length and unselect M sequences)?
+
+Based on the plot Sequence Counts, do all the samples have the 
+same number of reads? Are most of the reads unique?
+
+Based on the plot Sequence Quality Histograms, is the base quality
+good all along the reads? 
+
+

Step 4. Remove primers with Cutadapt

+

Select zippedFastq.tar and select the tool +Microbial amplicon data preprocessing for ASV / Remove primers and adapters with Cutadapt.

+

Click Parameters and set +Is the data paired end or single end reads = single.
+Give the primer sequences:

+
    +
  • Forward primer (in this case an M13 linker and the forward +primer):
    +The 5' adapter = TGTAAAACGACGGCCAGTGTCAGCTCGTGYYGTGAG
    +
  • +
  • Reverse primer: The 3' adapter = TTGYACACACCGCCCGT +(reverse complement of the reverse primer)
  • +
+

Also set Remove reads which were not trimmed = yes.

+

Close the parameter window and run the tool

+
Check the Cutadapt output (report.txt).   
+
+Look at the first sample and check how many times the forward primer (Adapter 1)  
+and reverse primer (Adapter 2) were trimmed.  
+
+Does Cutadapt with these settings detect exact matches of the primers?
+

Step 5. Trim sequences for base quality and sequence length +with Trimmomatic

+

Choose adapters_removed.tar and select the tool +Preprocessing / Trim Ion Torrent reads reads with Trimmomatic. +Set Sliding window trimming parameters = 10:20. Here we are +telling Trimmomatic to slide along each sequence read in stretches of 10 +bases, and if the average quality score of the window is below 20, trim +the read at that point. Also set +Minimum length of reads to keep = 200 and run the tool.

+

Choose trimmed.tarand run the MultiQC tool again +(Quality control / Read quality with MultiQC for many FASTQ files)

+
Do you see any differences in the sequence counts and sequence quality histograms   
+compared to the first time we ran the tool before primer removal and quality trimming?
+

Step 6. Combine FASTQ files and create count +file

+

Select trimmed.tar run the tool +Microbial amplicon data preprocessing for OTU / Combine FASTQ files into one FASTA file and make a Mothur count file.

+

Check the summary file sequences-summary.tsv.

+
How long are most of the sequences? 
+How long is the longest sequence?
+Are there any ambiguous bases in the sequences?
+How many sequences are there in total?
+

(Step 7. Screen sequences for ambiguous bases and +suspiciously long sequences)

+

Generally at this point, we would screen the sequences for example as +follows: Select sequences.fasta.gz and +sequences.count_table (use ctrl / cmd key to select +multiple files). Select the tool +Screen sequences for several criteria and in parameters set +Maximum number of ambiguous bases = 0 and +Maximum length = 400. However, we see from the output of +the previous step that these steps are not necessary for this data set +(there are no ambiguous bases and the longest sequence is only 333 bp), +so let’s skip this step and continue to step 8.

+

Step 8. Remove identical sequences

+

Select the files sequences.fasta.gzand +sequences.count_table (use ctrl / cmd key to select +multiple files) and run the tool +Extract unique sequences.

+

Note: If we had run step 7, we would have selected files +screened.fasta.gz and screened.count_table +here.

+

Check the summary file unique.summary.tsv.

+
How many unique sequences vs. total sequence are there?
+Why are we removing the identical sequences?
+

Step 9. Align sequences to reference

+

Next, we will align our sequences against the Silva v. 138.1 +reference alignment. Select the files unique.fasta.gz and +unique.count_table, and run the tool +Align sequences to reference so that you use only the +following section of the reference template alignment: +Start = 28500 and End = 43100.

+

(Note that these start and end values are selected for this specific +V6-V8 region amplicon. To modify the values for other regions, align an +example sequence (a small set of samples or the same fragment from E. +coli 16S rRNA gene) with the same tool and check where it is placed in +the reference.)

+

Open aligned-summary.tsv.

+
Where in the reference alignment do most of the sequences align? 
+Are there deviants?
+

Open also custom.reference.summary.tsv.

+
How long are the longest homopolymers in the reference? 
+How many sequences does the reference contain?
+

Step 10. Remove sequences which align outside the common +alignment range

+

Select aligned.fasta.gz and +unique.count_table, and the tool +Screen sequences for several criteria. Set +Alignment start position = 5963 (where most sequences start +based on aligned-summary.tsv), +Alignment end position = 12353 (before most sequences end), +and Maximum homopolymer length = 8. Run the tool.

+

Open summary.screened.tsv as spreadsheet.

+
How many unique sequences were removed (hint: compare with aligned-summary.tsv)? 
+How many sequences were removed overall?
+
+Were these sequences removed because: 
+1) they aligned outside the common alignment range, or 
+2) they contained too long homopolymers?
+

Step 11. Remove gaps and overhangs from the alignment. If +this creates new identical sequences, remove them

+

Choose screened.fasta.gz and +screened.count_table and run the tool +Filter sequence alignment.

+

Open filtered-log.txt.

+
How many columns we removed? How long is the alignment now? 
+
+Did the filtering generate new identical sequences, which were subsequently removed 
+(compare summary.screened.tsv and filtered-unique-summary.tsv)?
+

Step 12. Precluster the aligned sequences

+

Select filtered-unique.fasta.gz and +filtered-unique.count_table, and the tool +Precluster aligned sequences. Set +Number of differences allowed = 1 and run the tool.

+

Open preclustered-summary.tsv.

+
How many unique sequences do we have now?
+

Step 13. Remove chimeras

+

Select preclustered.fasta.gz and +preclustered.count_table and the tool +Remove chimeric sequences. Select +Reference = none, de novo, Method = vsearch +and Dereplicate = true and run the tool.

+

Open chimeras.removed.summary.tsv.

+
How many chimeras were removed?
+

Step 14. Classify sequences

+

Choose chimeras.removed.fasta.gz and +chimeras.removed.count_table, and run the tool +Classify 16S or 18S sequences to taxonomic units using Silva.

+

Open classification-summary.tsv and select Full Screen. +Sort the table by the taxon column (click on the word taxon).

+
Does your data contain chloroplast sequences? 
+

Step 15. Share your analysis session with a +colleague

+

Select your session in the session menu, click on the three dots, and +select Share. Create a new access rule: enter in the UserID field and set +Rights = read-only.

+
+
+ + + +
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+ + + + + + + + + + + + diff --git a/docs/IonTorrent/Exercises_day2.html b/docs/IonTorrent/Exercises_day2.html new file mode 100644 index 0000000..7d37ca2 --- /dev/null +++ b/docs/IonTorrent/Exercises_day2.html @@ -0,0 +1,1142 @@ + + + + + + + + + + Exercises_day2.knit + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
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+ + + + + + + + + + + + +
+
+

Day 2: Microbial community analysis

+
+

Getting the data into phyloseq

+

Step 16. Creating phyloseq input +files

+

Choose chimeras.removed.fasta.gz, +chimeras.removed.count_table and +sequences-taxonomy-assignment.txt. Next, run the tool +Microbial amplicon dta preprocessing for OTU / Generate input files for phyloseq +so that you select the correct data type (16S or 18S) and +set a cut-off of 0.03 (i.e. 3%, corresponding to 97% sequence +similarity) for OTU clustering.

+
Why are we using a dissimilarity threshold of 3%? 
+Can you think of examples where another threshold might be more appropriate?
+

Step 17. Filling in the phenodata file

+

The Generate input files for phyloseq tool produces a +phenodata file (a file type in Chipster used for documenting sample +groupings). Before proceeding with downstream analyses, let’s take a +moment to work on the phenodata.

+
    +
  1. Select the file file.opti_mcc.shared and in the +Selected files choose the tab Phenodata.

  2. +
  3. Remove the columns chiptype, group and +description by clicking the x. Create new +columns called site and bagging by clicking ++ Add column.

  4. +
  5. Check how the sample names in the column sample +start, and based on this enter the site codes HP and +KEK in the column site. Next, check the +characters after HPand KEK in the sample +names. When the next character is c, fill in control in the +column bagging. When the next characters are +ps, fill in bagged in the column +bagging.

  6. +
+

Check that your filled in phenodata table matches the example +below.

+

+

Step 18. Producing a phyloseq +object

+

Finally! 😄 We are done with pre-processing and ready to convert our +files into a phyloseq object that will be used in the +community analysis steps.

+

Select the Mothur shared file file.opti_mcc.shared and +constaxonomy file file.opti_mcc.0.03.cons.taxonomy. Select +the tool Convert Mothur files into phyloseq object. In +Parameters, specify the phenodata column including unique IDs +for each community profile (the column sample). Run the +tool.

+

This tool produces two files:

+
    +
  • a phyloseq object (stored as ps.Rda)
  • +
  • a text summary of the imported object +(ps_imported.txt).
  • +
+
Inspect the text summary a little closer. 
+What elements does the phyloseq object consist of?
+
+
+

Tidying and inspecting the data

+

Step 19. Data tidying

+
    +
  1. First, let’s remove any sequences that are not from our target +group Bacteria. Select ps.Rda and in the menu +Microbial amplicon data analyses the tool +Filter by taxonomic group tool. Choose +Bacteria as the group to retain. This helps ensure that we +only keep those data that are classified as Bacteria at the domain +level.

  2. +
  3. Earlier we already saw that our data contains chloroplast +sequences from plants, so let’s remove them together with any +mitochondrial sequences. Select the file ps_bacteria.Rda +and the tool Remove selected taxa. Set +Remove class Chloroplast = yes and +Remove family Mitochondria = yes and run the tool. You +could also use this tool to filter out other specific taxa such as known +contaminants.

  4. +
+
Why might you expect to see chloroplast or mitochondrial 
+sequences in a bacterial dataset - isn't that a little strange?
+

There are a few more additional tools for data tidying. Let’s first +get an overview of the distribution of OTUs in our data.

+
    +
  1. Selecting ps_ind.Rda, run the +Additional prevalence summaries tool. This will produce +both a prevalence plot (ps_prevalence.pdf) and a text +summary (ps_low.txt). The plot has a prevalence threshold +of 5% drawn as a default guess for prevalence filtering.
  2. +
+
How many doubletons are there in the data set?
+Do you have an advance idea about what the term "prevalence" refers to?
+Which bacterial groups are most common and abundant in the data?
+
    +
  1. There are two further tools for data tidying:
  2. +
+
    +
  • Remove OTUs with 0-2 occurrences
  • +
  • Proportional prevalence filtering (for removing OTUs that occur in +less than specific % of samples)
  • +
+

Selecting ps_ind.Rda, run the former tool, making sure +that both singletons and doubletons are removed.

+
Why would we want to remove singletons and doubletons from the data?
+Can you think of situations where these should be kept as part of the dataset?
+

Step 20. Sequence numbers, rarefaction curve and alpha +diversity indices

+

Select the file ps_ind.Rda. Try running the +Sequence numbers, rarefaction curve and alpha diversity estimates +tool. Choose a phenodata variable (site or +bagging) to tabulate the alpha diversity values.

+
Can you already make some basic inferences about the data 
+based on the rarefaction curve and alpha diversity values?
+
+Based on the rarefaction curve, what do you think might be 
+best - proceeding as is or rarefying the data to even depth?
+

Try the Rarefy OTU data to even depth tool using the +same file ps_ind.Rda. Select the resulting dataset +ps_rarefied.Rdaand and re-run the +Sequence numbers... tool.

+
Would you compare alpha diversity based on non-rarefied or rarefied data?
+
+
+

Taking a closer look at patterns

+

Step 21. Stacked taxon composition bar plots

+

At this point, we have many alternatives that depend on the study +goals. Let’s say that, in our case, the goal is to compare the overall +community structure between different sites and treatments (with +individual OTUs being of limited interest at this stage).

+

Nevertheless, first we probably want to visualise the data and +compare the sites and treatments in more detail. After all, we don’t +want to blindly rely on statistical test output!

+

Select ps_pruned.Rda and run the +Transform OTU counts tool, selecting +Relative abundances (%) as the data treatment. We can use +relative abundance data to produce bar plots.

+

This will produce a file called ps_relabund.Rda. Select +it and run the OTU relative abundance bar plots tool, +making sure that you have the Sample column selected in +Phenodata variable with sequencing sample IDs. There are +quite a few other parameters here that you can also modify. Feel free to +play around with the options but start with these:

+
    +
  • 1 in Relative abundance cut-off threshold (%) for excluding +OTUs
  • +
  • Class as the level of biological organisation
  • +
  • site as the phenodata variable 1 for plot faceting
  • +
+

The result should look close to this (click on the thumbnail to +expand the image):

+

+

Great! But let’s take a moment to think:

+
Based on the plot, what would you anticipate - are the sites likely to differ?
+Can you think of another way to visualise the data?
+

Step 22. Other ways to visualize the data

+

Going back to ps_pruned.Rda, let’s also visualise the +data using a multivariate ordination. For this, first use the tool +Transform OTU counts tool to perform a CLR transformation +(Centered log-ratio transformation with pseudocount).

+
Why might we want to use CLR transformation here, instead of % relative abundances?
+

Selecting the resulting file ps_clr.Rda, run the +Distance matrices and ordinations tool. Once again, one +must specify a column in the phenodata with unique IDs for each sample +(i.e. the Sample column). There are options for Euclidean +and Bray-Curtis distances - choose Euclidean and for the ordination +type, select nMDS. Also colour the ordination points by choosing +site in +Phenodata variable for grouping ordination points by colour +and define the shape by choosing bagging in +Phenodata variable for grouping ordination points by shape.

+

Here, we are using Aitchinson distances because we carried out a CLR +transformation and chose Euclidean distances. An alternative approach +would be to continue from the rarefied dataset +(ps_rarefied.Rda) and use Bray-Curtis dissimilarities to +produce the nMDS. In addition to nMDS, there is another ordination type +(db-RDA), but let’s focus on the nMDS for now.

+
Looking at the ordination (ps_ordiplot.pdf), what features stand out? 
+Does it look like any difference between groups could be due to a "location effect", 
+"dispersion effect" or both?
+
+What is the stress value of the nMDS (in ps_ordi.txt)? 
+Can you guess why this might be important?
+
+What is the advantage of plotting individual sample names here?
+

As a bonus topic, you might also want to produce a db-RDA plot and +consider this question:

+
When might you want to use db-RDA rather than nMDS?
+
+
+

Diving into statistics

+

Step 23. PERMANOVA

+

Ok! We’re ready to run some statistical tests now. Let’s proceed with +a permutational multivariate analysis of variance (PERMANOVA).

+

The Distance matrices and ordinations tool also produced +a distance matrix (ps_dist.Rda) that we can use for +statistical testing. Selecting it, run the +Global PERMANOVA for OTU abundance data tool, selecting +Main effects only as the PERMANOVA formula and specifying +site as Phenodata variable 1.

+
What does the word "global" mean here?
+
+How would you interpret the results (global_permanova.txt)? 
+Does the site explain community variation in our data?
+

Using ps_dist.Rda, also run the +PERMDISP for OTU abundance data tool, selecting the same +phenodata variable.

+
What does PERMDISP stand for? Why would we want to use it?
+
+Comparing the output of this test (permdisl.txt) with the global PERMANOVA results,  
+can you confirm whether the PERMANOVA result is due to a location or dispersion effect? 
+
+Does the result match your earlier anticipations based on inspecting the ordination?
+

There are also post-hoc pairwise tests for further community +comparisons. We do not need those for the present dataset, since we are +only comparing two groups at a time.

+

Step 24. DESeq2

+

If we were interested in OTU-level differences between sample groups, +we could turn to differential abundance analysis with DESeq2 instead of +PERMANOVA. For this, let’s return to the untransformed dataset +(ps_pruned.Rda).

+

Choosing that dataset and the Transform OTU counts tool, +select +DEseq2 format conversion and variance-stabilizing transformation +as the data transformation. For the DESeq2 format conversion, we also +need to specify a phenodata variable - for this, use site. +We get two files as a result:

+
    +
  • ps_vst.Rda
  • +
  • deseq2.Rda
  • +
+

Of these, deseq2.Rda can be used for DESeq2.

+

Selecting deseq2.Rda, run the +Differential OTU abundance analysis using DESeq2 tool, with +the number of sample groups set to 2, the lower-level taxonomic grouping +to Class and the higher-level grouping to Phylum.

+

Take a moment to inspect the results table +(deseq2_otutable.tsv) and results plot +(deseq2_otuplot.pdf).

+
Do some taxa seem more responsible for community-level differences than others?
+Which site is the reference level in the comparison, HP or KEK?
+Are  OTUs with positive log2 fold change values more abundant in HP or in KEK?
+
+
+
+ + + +
+ +
+
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