Importing demultiplexed sequence data
Contents
Importing demultiplexed sequence data¶
In this section of the tutorial, we’ll import raw fastq data that is already demultiplexed (i.e., separated into per-sample fastq files) into a QIIME 2 artifact.
Importing¶
We’ll begin with the data import.
import zipfile
url = 'https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import.zip'
fn = 'data_to_import.zip'
request.urlretrieve(url, fn)
with zipfile.ZipFile(fn) as zf:
zf.extractall('data_to_import')
wget \
-O 'data_to_import.zip' \
'https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import.zip'
unzip -d data_to_import data_to_import.zip
def casava_directory_factory():
import tempfile
import requests
import shutil
import qiime2
from q2_types.per_sample_sequences import \
CasavaOneEightSingleLanePerSampleDirFmt
sequence_data_url = 'https://data.qiime2.org/2022.2/tutorials/liao/fastq-casava.zip'
data = requests.get(sequence_data_url)
with tempfile.NamedTemporaryFile(mode='w+b') as f:
f.write(data.content)
f.flush()
dir_fmt = CasavaOneEightSingleLanePerSampleDirFmt()
shutil.unpack_archive(f.name, str(dir_fmt), 'zip')
return dir_fmt
data_to_import = use.init_format('data_to_import', casava_directory_factory)
- Using the
Upload Datatool: Steps to setup
data_to_import:sequences:On the fourth tab (Rule-based):
Set “Upload data as” to
Collection(s)Set “Load tabular data from” to
Pasted TablePaste the following contents into the large text area:
FMT.0093C_46_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093C_46_L001_R2_001.fastq.gz FMT.0093C_5_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093C_5_L001_R1_001.fastq.gz FMT.0093D_2_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093D_2_L001_R1_001.fastq.gz FMT.0093D_43_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093D_43_L001_R2_001.fastq.gz FMT.0093E_25_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093E_25_L001_R1_001.fastq.gz FMT.0093E_66_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093E_66_L001_R2_001.fastq.gz FMT.0093F_47_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093F_47_L001_R2_001.fastq.gz FMT.0093F_6_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093F_6_L001_R1_001.fastq.gz FMT.0093G_24_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093G_24_L001_R1_001.fastq.gz FMT.0093G_65_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093G_65_L001_R2_001.fastq.gz FMT.0093H_26_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093H_26_L001_R1_001.fastq.gz FMT.0093H_67_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093H_67_L001_R2_001.fastq.gz FMT.0093I_1_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093I_1_L001_R1_001.fastq.gz FMT.0093I_42_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093I_42_L001_R2_001.fastq.gz FMT.0093J_22_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093J_22_L001_R1_001.fastq.gz FMT.0093J_63_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093J_63_L001_R2_001.fastq.gz FMT.0093K_50_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093K_50_L001_R2_001.fastq.gz FMT.0093K_9_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093K_9_L001_R1_001.fastq.gz FMT.0093L_3_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093L_3_L001_R1_001.fastq.gz FMT.0093L_44_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093L_44_L001_R2_001.fastq.gz FMT.0093M_28_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093M_28_L001_R1_001.fastq.gz FMT.0093M_69_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093M_69_L001_R2_001.fastq.gz FMT.0093P_14_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093P_14_L001_R1_001.fastq.gz FMT.0093P_55_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093P_55_L001_R2_001.fastq.gz FMT.0093Q_39_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093Q_39_L001_R1_001.fastq.gz FMT.0093Q_80_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093Q_80_L001_R2_001.fastq.gz FMT.0093S_30_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093S_30_L001_R1_001.fastq.gz FMT.0093S_71_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093S_71_L001_R2_001.fastq.gz FMT.0093T_35_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093T_35_L001_R1_001.fastq.gz FMT.0093T_76_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093T_76_L001_R2_001.fastq.gz FMT.0093U_13_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093U_13_L001_R1_001.fastq.gz FMT.0093U_54_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093U_54_L001_R2_001.fastq.gz FMT.0093V_33_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093V_33_L001_R1_001.fastq.gz FMT.0093V_74_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093V_74_L001_R2_001.fastq.gz FMT.0093W_18_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093W_18_L001_R1_001.fastq.gz FMT.0093W_59_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093W_59_L001_R2_001.fastq.gz FMT.0093X_11_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093X_11_L001_R1_001.fastq.gz FMT.0093X_52_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0093X_52_L001_R2_001.fastq.gz FMT.0103V_27_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0103V_27_L001_R1_001.fastq.gz FMT.0103V_68_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0103V_68_L001_R2_001.fastq.gz FMT.0103W_0_L001_R1_001.fastq.gz 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https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0106I_64_L001_R2_001.fastq.gz FMT.0106L_21_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0106L_21_L001_R1_001.fastq.gz FMT.0106L_62_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0106L_62_L001_R2_001.fastq.gz FMT.0106M_45_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0106M_45_L001_R2_001.fastq.gz FMT.0106M_4_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0106M_4_L001_R1_001.fastq.gz FMT.0106N_29_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0106N_29_L001_R1_001.fastq.gz FMT.0106N_70_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0106N_70_L001_R2_001.fastq.gz FMT.0106R_38_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0106R_38_L001_R1_001.fastq.gz FMT.0106R_79_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0106R_79_L001_R2_001.fastq.gz FMT.0107B_15_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0107B_15_L001_R1_001.fastq.gz FMT.0107B_56_L001_R2_001.fastq.gz 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https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0107K_36_L001_R1_001.fastq.gz FMT.0107K_77_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0107K_77_L001_R2_001.fastq.gz FMT.0107L_31_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0107L_31_L001_R1_001.fastq.gz FMT.0107L_72_L001_R2_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0107L_72_L001_R2_001.fastq.gz FMT.0107M_16_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0107M_16_L001_R1_001.fastq.gz FMT.0107M_57_L001_R2_001.fastq.gz 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https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0107T_48_L001_R2_001.fastq.gz FMT.0107T_7_L001_R1_001.fastq.gz https://qiime2-workshops.s3.us-west-2.amazonaws.com/faes-jan2022/data/020-tutorial-upstream/030-importing/data_to_import/FMT.0107T_7_L001_R1_001.fastq.gz
Press the
buildbutton at the bottom.
In the resulting UI, do the following:
Add a rule by pressing the
+ Rulesbutton and choosingAdd / Modify Column Definitions.In the sidebar:
Press
+Add Definitionand selectList Identifier(s), then select columnA.Press
+Add Definitionand selectURL.Change the dropdown above the button to be
B. (You should see the table headers listA (List Identifier)andB (URL).)Press the
Applybutton.
In the bottom right, set “Name” to be
data_to_import:sequencesPress the
Uploadbutton at the bottom right.
data_to_import.zip| download
from q2_types.per_sample_sequences import CasavaOneEightSingleLanePerSampleDirFmt
from qiime2 import Artifact
demultiplexed_sequences = Artifact.import_data(
'SampleData[PairedEndSequencesWithQuality]',
'data_to_import',
CasavaOneEightSingleLanePerSampleDirFmt,
)
qiime tools import \
--type 'SampleData[PairedEndSequencesWithQuality]' \
--input-format CasavaOneEightSingleLanePerSampleDirFmt \
--input-path data_to_import \
--output-path demultiplexed-sequences.qza
from q2_types.per_sample_sequences import \
CasavaOneEightSingleLanePerSampleDirFmt
demultiplexed_sequences = use.import_from_format(
'demultiplexed_sequences',
semantic_type='SampleData[PairedEndSequencesWithQuality]',
variable=data_to_import,
view_type=CasavaOneEightSingleLanePerSampleDirFmt)
- Using the
qiime2 tools importtool: Set “Type of data to import” to
SampleData[PairedEndSequencesWithQuality]Set “QIIME 2 file format to import from” to
Casava One Eight Single Lane Per Sample Directory FormatFor
import_sequences, do the following:Leave “Select a mechanism” as
Use collection to importSet “elements” to
#: data_to_import:sequencesLeave “Append an extension?” as
No.
Press the
Executebutton.
- Once completed, for the new entry in your history, use the
Editbutton to set the name as follows: (Renaming is optional, but it will make any subsequent steps easier to complete.)
History Name
“Name” to set (be sure to press
Save)#: qiime2 tools import [...]demultiplexed-sequences.qza
Generating and viewing a summary of the imported data¶
After the import is complete, you can generate a summary of the imported artifact. This summary contains several important pieces of information.
First, it tells you how many sequences were obtained for each of the samples. The expected number of sequences per sample will vary depending on the sequencing technology that was applied and the the number of samples that were multiplexed in your run. You should review this, and ensure that you are getting the expected number of sequences on average.
Second, this summary provides interactive figures that illustrate sequence quality. This will give you an overview of the quality of your sequencing run, and you’ll need to extract information from these plots to perform quality control on the data in the next step of the tutorial.
import qiime2.plugins.demux.actions as demux_actions
demultiplexed_sequences_summ_viz, = demux_actions.summarize(
data=demultiplexed_sequences,
)
qiime demux summarize \
--i-data demultiplexed-sequences.qza \
--o-visualization demultiplexed-sequences-summ.qzv
use.action(
use.UsageAction(plugin_id='demux', action_id='summarize'),
use.UsageInputs(data=demultiplexed_sequences),
use.UsageOutputNames(visualization='demultiplexed_sequences_summ'),
)
- Using the
qiime2 demux summarizetool: Set “data” to
#: demultiplexed-sequences.qzaPress the
Executebutton.
- Once completed, for the new entry in your history, use the
Editbutton to set the name as follows: (Renaming is optional, but it will make any subsequent steps easier to complete.)
History Name
“Name” to set (be sure to press
Save)#: qiime2 demux summarize [...] : visualization.qzvdemultiplexed-sequences-summ.qzv