Amplicon worflow example using Dada2 and Phyloseq
This is a first draft of an Amplicon sequencing turorual for a planned ARS Microbiome workshop. It is modified from the Dada2 tutorial created by Benjamin Callahan, the Author of Dada2 with permission. https://benjjneb.github.io/dada2/tutorial.html
modified by Adam Rivers
Here we walk through version 1.4 of the DADA2 pipeline on a small multi-sample dataset. Our starting point is a set of Illumina-sequenced paired-end fastq files that have been split (or “demultiplexed”) by sample and from which the barcodes/adapters have already been removed. The end product is a sequence variant (SV) table, a higher-resolution analogue of the ubiquitous “OTU table”, which records the number of times each ribosomal sequence variant (SV) was observed in each sample. We also assign taxonomy to the output sequences, and demonstrate how the data can be imported into the popular phyloseq R package for the analysis of microbiome data.
Starting point
This workflow assumes that the data you are starting with meets certain criteria:
Non-biological nucleotides have been removed (primers/adapters/barcodes…) Samples are demultiplexed (split into individual per-sample fastqs) If paired-end sequencing, the forward and reverse fastqs contain reads in matched order If these criteria are not true for your data (are you sure there aren’t any primers hanging around?) you need to remedy those issues before beginning this workflow. See the FAQ for some recommendations for common issues.
Getting ready First we load the dada2 library. If you don’t already have the dada2 package, see the dada2 installation instructions
library(dada2); packageVersion("dada2")[1] ‘1.4.0’If your dada2 version shouldbe 1.4 or higher.
The data we will work with are the same as those in the Mothur Miseq SOP walkthrough. Download the example data and unzip. These files represent longitudinal samples from a mouse post-weaning and one mock community control. For now just consider them paired-end fastq files to be processed. Define the following path variable so that it points to the extracted directory on your machine:
This example uses data from
library("dada2")
path <- "/Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP"
list.files(path) [1] "F3D0_S188_L001_R1_001.fastq" "F3D0_S188_L001_R2_001.fastq"
[3] "F3D1_S189_L001_R1_001.fastq" "F3D1_S189_L001_R2_001.fastq"
[5] "F3D141_S207_L001_R1_001.fastq" "F3D141_S207_L001_R2_001.fastq"
[7] "F3D142_S208_L001_R1_001.fastq" "F3D142_S208_L001_R2_001.fastq"
[9] "F3D143_S209_L001_R1_001.fastq" "F3D143_S209_L001_R2_001.fastq"
[11] "F3D144_S210_L001_R1_001.fastq" "F3D144_S210_L001_R2_001.fastq"
[13] "F3D145_S211_L001_R1_001.fastq" "F3D145_S211_L001_R2_001.fastq"
[15] "F3D146_S212_L001_R1_001.fastq" "F3D146_S212_L001_R2_001.fastq"
[17] "F3D147_S213_L001_R1_001.fastq" "F3D147_S213_L001_R2_001.fastq"
[19] "F3D148_S214_L001_R1_001.fastq" "F3D148_S214_L001_R2_001.fastq"
[21] "F3D149_S215_L001_R1_001.fastq" "F3D149_S215_L001_R2_001.fastq"
[23] "F3D150_S216_L001_R1_001.fastq" "F3D150_S216_L001_R2_001.fastq"
[25] "F3D2_S190_L001_R1_001.fastq" "F3D2_S190_L001_R2_001.fastq"
[27] "F3D3_S191_L001_R1_001.fastq" "F3D3_S191_L001_R2_001.fastq"
[29] "F3D5_S193_L001_R1_001.fastq" "F3D5_S193_L001_R2_001.fastq"
[31] "F3D6_S194_L001_R1_001.fastq" "F3D6_S194_L001_R2_001.fastq"
[33] "F3D7_S195_L001_R1_001.fastq" "F3D7_S195_L001_R2_001.fastq"
[35] "F3D8_S196_L001_R1_001.fastq" "F3D8_S196_L001_R2_001.fastq"
[37] "F3D9_S197_L001_R1_001.fastq" "F3D9_S197_L001_R2_001.fastq"
[39] "HMP_MOCK.v35.fasta" "Mock_S280_L001_R1_001.fastq"
[41] "Mock_S280_L001_R2_001.fastq" "mouse.dpw.metadata"
[43] "mouse.time.design" "stability.batch"
[45] "stability.files" If the package successfully loaded and your listed files match those here, you are ready to go through the DADA2 pipeline.
Filter and Trim
# Sort ensures forward/reverse reads are in same order
fnFs <- sort(list.files(path, pattern="_R1_001.fastq"))
fnRs <- sort(list.files(path, pattern="_R2_001.fastq"))
# Extract sample names, assuming filenames have format: SAMPLENAME_XXX.fastq
sample.names <- sapply(strsplit(fnFs, "_"), `[`, 1)
# Specify the full path to the fnFs and fnRs
fnFs <- file.path(path, fnFs)
fnRs <- file.path(path, fnRs)If using this workflow on your own data: The string manipulations may have to be modified, especially the extraction of sample names from the file names.
Examine quality profiles of forward and reverse reads It is important to look at your data. We start by visualizing the quality profiles of the forward reads:
plotQualityProfile(fnFs[1:2])
The forward reads are good quality. We generally advise trimming the last few nucleotides to avoid less well-controlled errors that can arise there. There is no suggestion from these quality profiles that any additional trimming is needed, so we will truncate the forward reads at position 240 (trimming the last 10 nucleotides).
Now we visualize the quality profile of the reverse reads:
plotQualityProfile(fnRs[1:2])
The reverse reads are significantly worse quality, especially at the end, which is common in Illumina sequencing. This isn’t too worrisome, DADA2 incorporates quality information into its error model which makes the algorithm robust to lower quality sequence, but trimming as the average qualities crash is still a good idea as long as our reads will still overlap. We will truncate at position 160 where the quality distribution crashes.
If using this workflow on your own data: Your reads must overlap after truncation in order to merge them later!!! The tutorial is using 2x250 V4 sequence data, so the forward and reverse reads almost completely overlap and our trimming can be completely guided by the quality scores. If you are using a less-overlapping primer set, like V1-V2 or V3-V4, your truncLen must be large enough to maintain the overlap between them (the more the better).
Perform filtering and trimming
We define the filenames for the filtered fastq.gz files:
filt_path <- file.path(path, "filtered") # Place filtered files in filtered/ subdirectory
filtFs <- file.path(filt_path, paste0(sample.names, "_F_filt.fastq.gz"))
filtRs <- file.path(filt_path, paste0(sample.names, "_R_filt.fastq.gz"))We’ll use standard filtering parameters: maxN=0 (DADA2 requires no Ns), truncQ=2, rm.phix=TRUE and maxEE=2. The maxEE parameter sets the maximum number of “expected errors” allowed in a read, which is a better filter than simply averaging quality scores.
Filter the forward and reverse reads:
out <- filterAndTrim(fnFs, filtFs, fnRs, filtRs, truncLen=c(240,160),
maxN=0, maxEE=c(2,2), truncQ=2, rm.phix=TRUE,
compress=TRUE, multithread=TRUE)Creating output directory:/Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filteredhead(out) reads.in reads.out
F3D0_S188_L001_R1_001.fastq 7793 7113
F3D1_S189_L001_R1_001.fastq 5869 5299
F3D141_S207_L001_R1_001.fastq 5958 5463
F3D142_S208_L001_R1_001.fastq 3183 2914
F3D143_S209_L001_R1_001.fastq 3178 2941
F3D144_S210_L001_R1_001.fastq 4827 4312If using this workflow on your own data: The standard filtering parameters are starting points, not set in stone. For example, if too few reads are passing the filter, considering relaxing maxEE, perhaps especially on the reverse reads (eg. maxEE=c(2,5)). If you want to speed up downstream computation, consider tightening maxEE. For pair-end reads consider the length of your amplicon when choosing truncLen as your reads must overlap after truncation in order to merge them later!!!
If using this workflow on your own data: For common ITS amplicon strategies, it is undesirable to truncate reads to a fixed length due to the large amount of length variation at that locus. That is OK, just leave out truncLen. Make sure you removed the forward and reverse primers from both the forward and reverse reads though!
# Learn the Error Rates The DADA2 algorithm depends on a parametric error model (err) and every amplicon dataset has a different set of error rates. The learnErrors method learns the error model from the data, by alternating estimation of the error rates and inference of sample composition until they converge on a jointly consistent solution. As in many optimization problems, the algorithm must begin with an initial guess, for which the maximum possible error rates in this data are used (the error rates if only the most abundant sequence is correct and all the rest are errors).
The following runs in about 1.5 minutes on a 2016 Macbook Pro:
# Start the clock
ptm <- proc.time()
# Learn error rates
errF <- learnErrors(filtFs, multithread=TRUE)Initializing error rates to maximum possible estimate.
Sample 1 - 7113 reads in 1979 unique sequences.
Sample 2 - 5299 reads in 1639 unique sequences.
Sample 3 - 5463 reads in 1477 unique sequences.
Sample 4 - 2914 reads in 904 unique sequences.
Sample 5 - 2941 reads in 939 unique sequences.
Sample 6 - 4312 reads in 1267 unique sequences.
Sample 7 - 6741 reads in 1756 unique sequences.
Sample 8 - 4560 reads in 1438 unique sequences.
Sample 9 - 15637 reads in 3590 unique sequences.
Sample 10 - 11413 reads in 2762 unique sequences.
Sample 11 - 12017 reads in 3021 unique sequences.
Sample 12 - 5032 reads in 1566 unique sequences.
Sample 13 - 18075 reads in 3707 unique sequences.
Sample 14 - 6250 reads in 1479 unique sequences.
Sample 15 - 4052 reads in 1195 unique sequences.
Sample 16 - 7369 reads in 1832 unique sequences.
Sample 17 - 4765 reads in 1183 unique sequences.
Sample 18 - 4871 reads in 1382 unique sequences.
Sample 19 - 6504 reads in 1709 unique sequences.
Sample 20 - 4314 reads in 897 unique sequences.
selfConsist step 2
selfConsist step 3
selfConsist step 4
selfConsist step 5
Convergence after 5 rounds.
Total reads used: 139642 # Stop the clock
proc.time() - ptm user system elapsed
204.395 4.690 82.523 # Start the clock
ptm <- proc.time()
# Learn error rates
errR <- learnErrors(filtRs, multithread=TRUE)Initializing error rates to maximum possible estimate.
Sample 1 - 7113 reads in 1660 unique sequences.
Sample 2 - 5299 reads in 1349 unique sequences.
Sample 3 - 5463 reads in 1335 unique sequences.
Sample 4 - 2914 reads in 853 unique sequences.
Sample 5 - 2941 reads in 880 unique sequences.
Sample 6 - 4312 reads in 1286 unique sequences.
Sample 7 - 6741 reads in 1803 unique sequences.
Sample 8 - 4560 reads in 1265 unique sequences.
Sample 9 - 15637 reads in 3414 unique sequences.
Sample 10 - 11413 reads in 2522 unique sequences.
Sample 11 - 12017 reads in 2771 unique sequences.
Sample 12 - 5032 reads in 1415 unique sequences.
Sample 13 - 18075 reads in 3290 unique sequences.
Sample 14 - 6250 reads in 1390 unique sequences.
Sample 15 - 4052 reads in 1134 unique sequences.
Sample 16 - 7369 reads in 1635 unique sequences.
Sample 17 - 4765 reads in 1084 unique sequences.
Sample 18 - 4871 reads in 1161 unique sequences.
Sample 19 - 6504 reads in 1502 unique sequences.
Sample 20 - 4314 reads in 732 unique sequences.
selfConsist step 2
selfConsist step 3
selfConsist step 4
selfConsist step 5
selfConsist step 6
Convergence after 6 rounds.
Total reads used: 139642 # Stop the clock
proc.time() - ptm user system elapsed
163.384 4.410 66.595 It is always worthwhile, as a sanity check if nothing else, to visualize the estimated error rates:
plotErrors(errF, nominalQ=TRUE)
The error rates for each possible transition (eg. A->C, A->G, …) are shown. Points are the observed error rates for each consensus quality score. The black line shows the estimated error rates after convergence. The red line shows the error rates expected under the nominal definition of the Q-value. Here the black line (the estimated rates) fits the observed rates well, and the error rates drop with increased quality as expected. Everything looks reasonable and we proceed with confidence.
If using this workflow on your own data: Parameter learning is computationally intensive, so by default the learnErrors function uses only a subset of the data (the first 1M reads). If the plotted error model does not look like a good fit, try increasing the nreads parameter to see if the fit improves.
Dereplication
Dereplication combines all identical sequencing reads into into “unique sequences” with a corresponding “abundance”: the number of reads with that unique sequence. Dereplication substantially reduces computation time by eliminating redundant comparisons.
Dereplication in the DADA2 pipeline has one crucial addition from other pipelines: DADA2 retains a summary of the quality information associated with each unique sequence. The consensus quality profile of a unique sequence is the average of the positional qualities from the dereplicated reads. These quality profiles inform the error model of the subsequent denoising step, significantly increasing DADA2’s accuracy.
Dereplicate the filtered fastq files:
derepFs <- derepFastq(filtFs, verbose=TRUE)Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D0_F_filt.fastq.gz
Encountered 1979 unique sequences from 7113 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D1_F_filt.fastq.gz
Encountered 1639 unique sequences from 5299 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D141_F_filt.fastq.gz
Encountered 1477 unique sequences from 5463 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D142_F_filt.fastq.gz
Encountered 904 unique sequences from 2914 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D143_F_filt.fastq.gz
Encountered 939 unique sequences from 2941 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D144_F_filt.fastq.gz
Encountered 1267 unique sequences from 4312 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D145_F_filt.fastq.gz
Encountered 1756 unique sequences from 6741 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D146_F_filt.fastq.gz
Encountered 1438 unique sequences from 4560 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D147_F_filt.fastq.gz
Encountered 3590 unique sequences from 15637 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D148_F_filt.fastq.gz
Encountered 2762 unique sequences from 11413 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D149_F_filt.fastq.gz
Encountered 3021 unique sequences from 12017 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D150_F_filt.fastq.gz
Encountered 1566 unique sequences from 5032 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D2_F_filt.fastq.gz
Encountered 3707 unique sequences from 18075 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D3_F_filt.fastq.gz
Encountered 1479 unique sequences from 6250 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D5_F_filt.fastq.gz
Encountered 1195 unique sequences from 4052 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D6_F_filt.fastq.gz
Encountered 1832 unique sequences from 7369 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D7_F_filt.fastq.gz
Encountered 1183 unique sequences from 4765 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D8_F_filt.fastq.gz
Encountered 1382 unique sequences from 4871 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D9_F_filt.fastq.gz
Encountered 1709 unique sequences from 6504 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/Mock_F_filt.fastq.gz
Encountered 897 unique sequences from 4314 total sequences read.derepRs <- derepFastq(filtRs, verbose=TRUE)Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D0_R_filt.fastq.gz
Encountered 1660 unique sequences from 7113 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D1_R_filt.fastq.gz
Encountered 1349 unique sequences from 5299 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D141_R_filt.fastq.gz
Encountered 1335 unique sequences from 5463 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D142_R_filt.fastq.gz
Encountered 853 unique sequences from 2914 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D143_R_filt.fastq.gz
Encountered 880 unique sequences from 2941 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D144_R_filt.fastq.gz
Encountered 1286 unique sequences from 4312 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D145_R_filt.fastq.gz
Encountered 1803 unique sequences from 6741 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D146_R_filt.fastq.gz
Encountered 1265 unique sequences from 4560 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D147_R_filt.fastq.gz
Encountered 3414 unique sequences from 15637 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D148_R_filt.fastq.gz
Encountered 2522 unique sequences from 11413 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D149_R_filt.fastq.gz
Encountered 2771 unique sequences from 12017 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D150_R_filt.fastq.gz
Encountered 1415 unique sequences from 5032 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D2_R_filt.fastq.gz
Encountered 3290 unique sequences from 18075 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D3_R_filt.fastq.gz
Encountered 1390 unique sequences from 6250 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D5_R_filt.fastq.gz
Encountered 1134 unique sequences from 4052 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D6_R_filt.fastq.gz
Encountered 1635 unique sequences from 7369 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D7_R_filt.fastq.gz
Encountered 1084 unique sequences from 4765 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D8_R_filt.fastq.gz
Encountered 1161 unique sequences from 4871 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/F3D9_R_filt.fastq.gz
Encountered 1502 unique sequences from 6504 total sequences read.
Dereplicating sequence entries in Fastq file: /Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/MiSeq_SOP/filtered/Mock_R_filt.fastq.gz
Encountered 732 unique sequences from 4314 total sequences read.# Name the derep-class objects by the sample names
names(derepFs) <- sample.names
names(derepRs) <- sample.namesIf using this workflow on your own data: The tutorial dataset is small enough to easily load into memory. If your dataset exceeds available RAM, it is preferable to process samples one-by-one in a streaming fashion: see the DADA2 Workflow on Big Data for an example.
Sample Inference
We are now ready to apply the core sequence-variant inference algorithm to the dereplicated data.
Infer the sequence variants in each sample:
# Start the clock
ptm <- proc.time()
dadaFs <- dada(derepFs, err=errF, multithread=TRUE)Sample 1 - 7113 reads in 1979 unique sequences.
Sample 2 - 5299 reads in 1639 unique sequences.
Sample 3 - 5463 reads in 1477 unique sequences.
Sample 4 - 2914 reads in 904 unique sequences.
Sample 5 - 2941 reads in 939 unique sequences.
Sample 6 - 4312 reads in 1267 unique sequences.
Sample 7 - 6741 reads in 1756 unique sequences.
Sample 8 - 4560 reads in 1438 unique sequences.
Sample 9 - 15637 reads in 3590 unique sequences.
Sample 10 - 11413 reads in 2762 unique sequences.
Sample 11 - 12017 reads in 3021 unique sequences.
Sample 12 - 5032 reads in 1566 unique sequences.
Sample 13 - 18075 reads in 3707 unique sequences.
Sample 14 - 6250 reads in 1479 unique sequences.
Sample 15 - 4052 reads in 1195 unique sequences.
Sample 16 - 7369 reads in 1832 unique sequences.
Sample 17 - 4765 reads in 1183 unique sequences.
Sample 18 - 4871 reads in 1382 unique sequences.
Sample 19 - 6504 reads in 1709 unique sequences.
Sample 20 - 4314 reads in 897 unique sequences.# Stop the clock
proc.time() - ptm user system elapsed
44.297 0.835 16.595 dadaRs <- dada(derepRs, err=errR, multithread=TRUE)Sample 1 - 7113 reads in 1660 unique sequences.
Sample 2 - 5299 reads in 1349 unique sequences.
Sample 3 - 5463 reads in 1335 unique sequences.
Sample 4 - 2914 reads in 853 unique sequences.
Sample 5 - 2941 reads in 880 unique sequences.
Sample 6 - 4312 reads in 1286 unique sequences.
Sample 7 - 6741 reads in 1803 unique sequences.
Sample 8 - 4560 reads in 1265 unique sequences.
Sample 9 - 15637 reads in 3414 unique sequences.
Sample 10 - 11413 reads in 2522 unique sequences.
Sample 11 - 12017 reads in 2771 unique sequences.
Sample 12 - 5032 reads in 1415 unique sequences.
Sample 13 - 18075 reads in 3290 unique sequences.
Sample 14 - 6250 reads in 1390 unique sequences.
Sample 15 - 4052 reads in 1134 unique sequences.
Sample 16 - 7369 reads in 1635 unique sequences.
Sample 17 - 4765 reads in 1084 unique sequences.
Sample 18 - 4871 reads in 1161 unique sequences.
Sample 19 - 6504 reads in 1502 unique sequences.
Sample 20 - 4314 reads in 732 unique sequences.Inspecting the dada-class object returned by dada:
dadaFs[[1]]dada-class: object describing DADA2 denoising results
128 sample sequences were inferred from 1979 input unique sequences.
Key parameters: OMEGA_A = 1e-40, BAND_SIZE = 16, USE_QUALS = TRUEThe DADA2 algorithm inferred 128 real variants from the 1979 unique sequences in the first sample. There is much more to the dada-class return object than this (see help(“dada-class”) for some info), including multiple diagnostics about the quality of each inferred sequence variant, but that is beyond the scope of an introductory tutorial.
If using this workflow on your own data: All samples are simultaneously loaded into memory in the tutorial. If you are dealing with datasets that approach or exceed available RAM, it is preferable to process samples one-by-one in a streaming fashion: see the DADA2 Workflow on Big Data for an example.
If using this workflow on your own data: By default, the dada function processes each sample independently, but pooled processing is available with pool=TRUE and that may give better results for low sampling depths at the cost of increased computation time. See our discussion about pooling samples for sample inference.
Merge paired reads
Spurious sequence variants are further reduced by merging overlapping reads. The core function here is mergePairs, which depends on the forward and reverse reads being in matching order at the time they were dereplicated.
Merge the denoised forward and reverse reads:
mergers <- mergePairs(dadaFs, derepFs, dadaRs, derepRs, verbose=TRUE)6600 paired-reads (in 105 unique pairings) successfully merged out of 7113 (in 254 pairings) input.
5078 paired-reads (in 100 unique pairings) successfully merged out of 5299 (in 192 pairings) input.
5047 paired-reads (in 78 unique pairings) successfully merged out of 5463 (in 199 pairings) input.
2663 paired-reads (in 52 unique pairings) successfully merged out of 2914 (in 142 pairings) input.
2575 paired-reads (in 54 unique pairings) successfully merged out of 2941 (in 162 pairings) input.
3668 paired-reads (in 53 unique pairings) successfully merged out of 4312 (in 203 pairings) input.
6202 paired-reads (in 81 unique pairings) successfully merged out of 6741 (in 230 pairings) input.
4040 paired-reads (in 90 unique pairings) successfully merged out of 4560 (in 233 pairings) input.
14340 paired-reads (in 142 unique pairings) successfully merged out of 15637 (in 410 pairings) input.
10599 paired-reads (in 117 unique pairings) successfully merged out of 11413 (in 331 pairings) input.
11197 paired-reads (in 134 unique pairings) successfully merged out of 12017 (in 342 pairings) input.
4426 paired-reads (in 83 unique pairings) successfully merged out of 5032 (in 233 pairings) input.
17477 paired-reads (in 148 unique pairings) successfully merged out of 18075 (in 330 pairings) input.
5907 paired-reads (in 80 unique pairings) successfully merged out of 6250 (in 193 pairings) input.
3770 paired-reads (in 85 unique pairings) successfully merged out of 4052 (in 194 pairings) input.
6915 paired-reads (in 98 unique pairings) successfully merged out of 7369 (in 226 pairings) input.
4480 paired-reads (in 66 unique pairings) successfully merged out of 4765 (in 159 pairings) input.
4606 paired-reads (in 96 unique pairings) successfully merged out of 4871 (in 204 pairings) input.
6173 paired-reads (in 108 unique pairings) successfully merged out of 6504 (in 196 pairings) input.
4279 paired-reads (in 20 unique pairings) successfully merged out of 4314 (in 33 pairings) input.# Inspect the merger data.frame from the first sample
head(mergers[[1]])We now have a data.frame for each sample with the merged $sequence, its $abundance, and the indices of the merged $forward and $reverse denoised sequences. Paired reads that did not exactly overlap were removed by mergePairs.
If using this workflow on your own data: Most of your reads should successfully merge. If that is not the case upstream parameters may need to be revisited: Did you trim away the overlap between your reads?
Construct sequence table We can now construct a “sequence table” of our mouse samples, a higher-resolution version of the “OTU table” produced by classical methods:
seqtab <- makeSequenceTable(mergers)The sequences being tabled vary in length.dim(seqtab)# Inspect distribution of sequence lengths
table(nchar(getSequences(seqtab)))
251 252 253 254 255
1 87 192 6 2 hist(nchar(getSequences(seqtab)), main="Distribution of sequence lengths")
The sequence table is a matrix with rows corresponding to (and named by) the samples, and columns corresponding to (and named by) the sequence variants. The lengths of our merged sequences all fall within the expected range for this V4 amplicon.
If using this workflow on your own data: Sequences that are much longer or shorter than expected may be the result of non-specific priming, and may be worth removing (eg. seqtab2 <- seqtab[,nchar(colnames(seqtab)) %in% seq(250,256)]). This is analogous to “cutting a band” in-silico to get amplicons of the targeted length.
Remove chimeras
The core dada method removes substitution and indel errors, but chimeras remain. Fortunately, the accuracy of the sequences after denoising makes identifying chimeras simpler than it is when dealing with fuzzy OTUs: all sequences which can be exactly reconstructed as a bimera (two-parent chimera) from more abundant sequences.
seqtab.nochim <- removeBimeraDenovo(seqtab, method="consensus", multithread=TRUE, verbose=TRUE)Identified 59 bimeras out of 288 input sequences.dim(seqtab.nochim)[1] 20 229sum(seqtab.nochim)/sum(seqtab)[1] 0.9643192The fraction of chimeras varies based on factors including experimental procedures and sample complexity, but can be substantial. Here chimeras make up about 20% of the inferred sequence variants, but those variants account for only about 4% of the total sequence reads.
If using this workflow on your own data: Most of your reads should remain after chimera removal (it is not uncommon for a majority of sequence variants to be removed though). If most of your reads were removed as chimeric, upstream processing may need to be revisited. In almost all cases this is caused by primer sequences with ambiguous nucleotides that were not removed prior to beginning the DADA2 pipeline.
Track reads through the pipeline
As a final check of our progress, we’ll look at the number of reads that made it through each step in the pipeline:
getN <- function(x) sum(getUniques(x))
track <- cbind(out, sapply(dadaFs, getN), sapply(mergers, getN), rowSums(seqtab), rowSums(seqtab.nochim))
colnames(track) <- c("input", "filtered", "denoised", "merged", "tabled", "nonchim")
rownames(track) <- sample.names
head(track) input filtered denoised merged tabled nonchim
F3D0 7793 7113 7113 6600 6600 6588
F3D1 5869 5299 5299 5078 5078 5067
F3D141 5958 5463 5463 5047 5047 4928
F3D142 3183 2914 2914 2663 2663 2600
F3D143 3178 2941 2941 2575 2575 2550
F3D144 4827 4312 4312 3668 3668 3527Looks good, we kept the majority of our raw reads, and there is no over-large drop associated with any single step.
If using this workflow on your own data: This is a great place to do a last sanity check. Outside of filtering (depending on how stringent you want to be) there should no step in which a majority of reads are lost. If a majority of reads failed to merge, you may need to revisit the truncLen parameter used in the filtering step and make sure that the truncated reads span your amplicon. If a majority of reads failed to pass the chimera check, you may need to revisit the removal of primers, as the ambiguous nucleotides in unremoved primers interfere with chimera identification.
Assign taxonomy
It is common at this point, especially in 16S/18S/ITS amplicon sequencing, to classify sequence variants taxonomically. The DADA2 package provides a native implementation of the RDP’s naive Bayesian classifier for this purpose. The assignTaxonomy function takes a set of sequences and a training set of taxonomically classified sequences, and outputs the taxonomic assignments with at least minBoot bootstrap confidence.
Appropriately formatted training fastas for the RDP training set 14, the GreenGenes 13.8 release clustered at 97% identity, the Silva reference database v123 (Silva dual license), and the UNITE ITS database (use the General Fasta release files) are available. To follow along, download the silva_nr_v123_train_set.fa.gz file, and place it in the directory with the fastq files.
The following databases are available: * Maintained: * GreenGenes version 13.8 * RDP version 14 * Silva version 123 (Silva dual-license) * UNITE (General Fasta releases) (version 1.3.3 or later of the dada2 package) * Contributed: * HitDB version 1 (Human InTestinal 16S rRNA)
Note that currently species-assignment training fastas are only available for the Silva and RDP databases. In addition to thanking the folks at RDP, Silva and GreenGenes for making these datasets available, we also want to thank Pat Schloss and the mothur team for making cleaner versions of the Silva and RDP training set available. To be specific, we created the dada2-compatible training fastas from the mothur-compatible Silva.nr_v123 files (described here, and license here), the mothur-compatible 16S rRNA reference (RDP) (described here), and the GreenGenes 13.8 OTUs clustered at 97%.
Formatting custom databases
Custom databases can be used as well, provided they can be converted to the dada2-compatible training fasta format.
The assignTaxonomy(…) function expects the training data to be provided in the form of a fasta file (or compressed fasta file) in which the taxonomy corresponding to each sequence is encoded in the id line in the following fashion (the second sequence is not assigned down to level 6):
taxtrain <- "/Users/rivers/Documents/Microbiome workshop/Amplicon tutorial/silva_nr_v123_train_set.fa.gz"
taxa <- assignTaxonomy(seqtab.nochim, taxtrain, multithread=TRUE)
unname(head(taxa)) [,1] [,2] [,3] [,4]
[1,] "Bacteria" "Bacteroidetes" "Bacteroidia" "Bacteroidales"
[2,] "Bacteria" "Bacteroidetes" "Bacteroidia" "Bacteroidales"
[3,] "Bacteria" "Bacteroidetes" "Bacteroidia" "Bacteroidales"
[4,] "Bacteria" "Bacteroidetes" "Bacteroidia" "Bacteroidales"
[5,] "Bacteria" "Bacteroidetes" "Bacteroidia" "Bacteroidales"
[6,] "Bacteria" "Bacteroidetes" "Bacteroidia" "Bacteroidales"
[,5] [,6]
[1,] "Bacteroidales_S24-7_group" NA
[2,] "Bacteroidales_S24-7_group" NA
[3,] "Bacteroidales_S24-7_group" NA
[4,] "Bacteroidales_S24-7_group" NA
[5,] "Bacteroidaceae" "Bacteroides"
[6,] "Bacteroidales_S24-7_group" NA Okay you’ve done it. You’ve sequenced, cleaned, clustered, removed chimeras and identified the microbial sequences in your sample. Now it’s time to begin making sense of that data.
Handoff to phyloseq
The DADA2 pipeline produced a sequence table and a taxonomy table which is appropriate for further analysis in phyloseq. We’ll also include the small amount of metadata we have – the samples are named by the gender (G), mouse subject number (X) and the day post-weaning (Y) it was sampled (eg. GXDY).
Import into phyloseq:
library(phyloseq); packageVersion("phyloseq")
Attaching package: ‘phyloseq’
The following object is masked from ‘package:SummarizedExperiment’:
distance
The following object is masked from ‘package:Biobase’:
sampleNames
The following object is masked from ‘package:GenomicRanges’:
distance
The following object is masked from ‘package:IRanges’:
distance[1] ‘1.20.0’library(ggplot2); packageVersion("ggplot2")[1] ‘2.2.1’# Make a data.frame holding the sample data
samples.out <- rownames(seqtab.nochim)
subject <- sapply(strsplit(samples.out, "D"), `[`, 1)
gender <- substr(subject,1,1)
subject <- substr(subject,2,999)
day <- as.integer(sapply(strsplit(samples.out, "D"), `[`, 2))
samdf <- data.frame(Subject=subject, Gender=gender, Day=day)
samdf$When <- "Early"
samdf$When[samdf$Day>100] <- "Late"
rownames(samdf) <- samples.out
# Construct phyloseq object (straightforward from dada2 outputs)
ps <- phyloseq(otu_table(seqtab.nochim, taxa_are_rows=FALSE),
sample_data(samdf),
tax_table(taxa))
ps <- prune_samples(sample_names(ps) != "Mock", ps) # Remove mock sample
psphyloseq-class experiment-level object
otu_table() OTU Table: [ 229 taxa and 19 samples ]
sample_data() Sample Data: [ 19 samples by 4 sample variables ]
tax_table() Taxonomy Table: [ 229 taxa by 6 taxonomic ranks ]Plot the species richness
plot_richness(ps, x="Day", measures=c("Shannon", "Simpson"), color="When") + theme_bw()The data you have provided does not have
any singletons. This is highly suspicious. Results of richness
estimates (for example) are probably unreliable, or wrong, if you have already
trimmed low-abundance taxa from the data.
We recommended that you find the un-trimmed data and retry.
No obvious systematic difference in alpha-diversity between early and late samples.
Create ordination plots
ord.nmds.bray <- ordinate(ps, method="NMDS", distance="bray")Square root transformation
Wisconsin double standardization
Run 0 stress 0.08908957
Run 1 stress 0.3747428
Run 2 stress 0.3382376
Run 3 stress 0.08908957
... New best solution
... Procrustes: rmse 9.56286e-06 max resid 2.118182e-05
... Similar to previous best
Run 4 stress 0.149361
Run 5 stress 0.1493614
Run 6 stress 0.09001141
Run 7 stress 0.1474737
Run 8 stress 0.09001141
Run 9 stress 0.1492644
Run 10 stress 0.08908957
... Procrustes: rmse 1.199293e-05 max resid 2.827523e-05
... Similar to previous best
Run 11 stress 0.09001141
Run 12 stress 0.08908957
... Procrustes: rmse 4.099192e-06 max resid 1.001481e-05
... Similar to previous best
Run 13 stress 0.09001141
Run 14 stress 0.08908957
... Procrustes: rmse 8.910287e-06 max resid 2.322558e-05
... Similar to previous best
Run 15 stress 0.09001141
Run 16 stress 0.1550556
Run 17 stress 0.09001141
Run 18 stress 0.08908957
... Procrustes: rmse 1.392618e-05 max resid 3.227656e-05
... Similar to previous best
Run 19 stress 0.156734
Run 20 stress 0.1474731
*** Solution reachedplot_ordination(ps, ord.nmds.bray, color="When", title="Bray NMDS")
Ordination picks out a clear separation between the early and late samples.
Bar plot
top20 <- names(sort(taxa_sums(ps), decreasing=TRUE))[1:20]
ps.top20 <- transform_sample_counts(ps, function(OTU) OTU/sum(OTU))
ps.top20 <- prune_taxa(top20, ps.top20)
plot_bar(ps.top20, x="Day", fill="Family") + facet_wrap(~When, scales="free_x")
Nothing glaringly obvious jumps out from the taxonomic distribution of the top 20 sequences to explain the early-late differentiation.
Phylogenetic trees of amplicon sequences
It is common to create a phylogenetic tree of the taxa and then use metrics like UNIFRAC distance or just plot datain a phylogentic context.
That can be done in phyloseq too.
Align the sequnces
library("msa")
seqs <- getSequences(seqtab.nochim)
names(seqs) <- seqs # This propagates to the tip labels of the tree
mult <- msa(seqs, method="ClustalW", type="dna", order="input")use default substitution matrixDODO
- Make tree
- Calculate unifrac with the tree
- place data on tree see: https://bioconductor.org/packages/release/bioc/vignettes/phyloseq/inst/doc/phyloseq-analysis.html https://f1000research.com/articles/5-1492/v2
- DESEQ2
- SPIEC-EASI networks