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Prepare PlateInfo & bcl2fastq SampleSheet

Prepare bcl2fastq sample sheet for demultiplexing illumina primer index

Related Commands

# 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.

What is the PlateInfo file?

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

# print V1 template
yap default-plate-info -v V1

# print V2 template
yap default-plate-info -v V2

PlateInfo Examples

  • This example contains four plates

  • Each plate is indexed by one PCR index

  • 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

Step 2: Run yap make-sample-sheet

  • 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.

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