# Print out template of the plate info
yap default-plate-info
# Make bcl2fastq sample sheet based on the plate info file
yap make-sample-sheet
Purpose
In this step, we prepare a PlateInfo file that contains sample metadata and PCR index information of the plates in your library. This information is used to generate a SampleSheet file for bcl2fastq to demultiplex the PCR index when generating raw FASTQ files.
Input
Library preparation information, most importantly:
The sample metadata (you determine what is necessary for your experiment)
The plate IDs of this library
The barcode information of this library
Step 1: Prepare a PlateInfo file
Get the PlateInfo file template and manually edit plate and barcode information
Primer names must be unique for the same sequencing run.
PlateInfo section must be Tab \t separated.
When demultiplexing, yap will automatically separate the single multiplex group into six, so during mapping, each plate will have six commands, the same as "V2 Index Library" on the next tab.
[CriticalInfo]
n_random_index=384
input_plate_size=384
pool_id=Pool_9
tube_label=Pool_9
email=your_email_address1@salk.edu;your_email_address2@salk.edu;your_email_address3@salk.edu
[LibraryInfo]
lib_comp_date=200518
project=DVC
organism=mm
dev_stage_age=P120
tissue_cell_type=VC
lib_type=snmCT-seq
sequencer=NovaSeq
se_pe=pe
read_length=150
requested_by=HL
[PlateInfo]
plate_id multiplex_group primer_name
DVC200116_P120_VC_B_M_1_4 1 D11
DVC200116_P120_VC_B_M_1_6 1 N6
DVC200116_P120_VC_B_M_1_8 1 K22
DVC200116_P120_VC_B_M_2_4 1 P8
# each plate is indexed by single PCR index, use multiplex_group 1 in this case
This example contains four plates
Each plate has six multiplex groups
All primer names must be unique for the same sequencing run.
Starting v1.3.5, If each plate in your library is indexed by a single PCR index, you can put each plate in a single row with multiplex_group = 1, the demultiplex pipeline will recognize it and automatically separate files into multiple multiplex groups.
This example contains eight plates
Every two plates share a primer quarter
Possible primer_quarter values are:
SetB_Q1, SetB_Q2, SetB_Q3, SetB_Q4
Set1_Q1, Set1_Q2, Set1_Q3, Set1_Q4
Each primer_quarter appears no more than twice for the same sequencing run.
yap make-sample-sheet take a V1 or V2 plate info file to generate a bcl2fastq sample sheet.
The sample sheet and name pattern are automatically generated so the pipeline can automatically parse cell information during and after mapping.
See usage in the command line yap make-sample-sheet -h
yap make-sample-sheet -h
usage: yap make-sample-sheet [-h] --plate_info_path PLATE_INFO_PATH
--output_prefix OUTPUT_PREFIX
[--header_path HEADER_PATH]
optional arguments:
-h, --help show this help message and exit
Required inputs:
--plate_info_path PLATE_INFO_PATH, -p PLATE_INFO_PATH
Path of the plate information file. (default: None)
--output_prefix OUTPUT_PREFIX, -o OUTPUT_PREFIX
Output prefix, will generate 2 sample sheets, 1 for
miseq, 1 for novaseq (default: None)
Optional inputs:
--header_path HEADER_PATH
Path to the sample sheet header that contains
sequencer info. Will use default if not provided.
(default: None)
Output
PlateInfo file recording library preparation information
Three sample sheets for bcl2fastq demultiplexing. One for MiSeq, two for NovaSeq (v1, or v1.5).
For libraries sequenced with illumina v1.5 S4 kit, the i5 barcodes in the sample sheet are reverse complemented.
Output after bcl2fastq
The raw FASTQ generated by bcl2fastq using SampleSheet generated in this step will contain several (8 in V1 and 64 in V2) cells' reads.
The random index occurs at the 5' of R1.
The PCR index is trimmed from both R1 and R2 and put into the read name.