SeqShop: Sequence Mapping and Assembly Practical, June 2014

Step 0: Login to the machine & setup environment

1. Login to the windows machine
   • The username/password for the Windows machine should be written on it
2. Open putty
   • Start->.....
3. In putty, login to seqshop-server.sph.umich.edu
   • Server name: seqshop-server.sph.umich.edu
   • Enter your provided username & password
4. To simplify commands/typing, we will setup an environment variable to point to the GotCloud directory.

export GC=/home/mktrost/seqshop/

GotCloud Alignment Pipeline

Input Files

Sequence Data Files : FASTQs

The FASTQ files are provided to you by those who did the sequencing.

For this tutorial, we will use FASTQs for 6 1000Genome samples

ls ${GC}/inputs/fastq/

There are 51 fastq files: combination of single-end & paired-end.

• Single-end: HG00641.chr7.CFTR.SRR069531.fastq
• Paired-end: HG00641.chr7.CFTR.SRR069531_1.fastq & HG00641.chr7.CFTR.SRR069531_2.fastq

Look at FASTQ:

less -S ${GC}/inputs/fastq/HG00641.chr7.CFTR.SRR069531.fastq

less is a Linux command that allows you to look at a file.

• -S option prevents line wrap.
• Use the arrow (up/down/left/right) keys to scroll through the file.
• Use zless if the file is compressed.

Reference Files

Reference files can be downloaded with GotCloud or from other sources.

ls ${GC}/reference/chr7

GotCloud FASTQ Index File

This file is created by you and directs GotCloud to your FASTQ files, providing additional information for them.

• tab delimited
• columns may be in any order
• starts with a header line
• one line per single-end read
• one line per paired-end read (only 1 line per pair).

Required Columns

Column Name	Description	Recommended Value
MERGE_NAME	Base name for the resulting BAM file for the sample Used to group multiple fastqs or fastq pairs into a single BAM	Sample Name
FASTQ1	Name of the fastq or the first in the pair if paired-end. (Only 1 line per pair)	path/fastq1
FASTQ2	Name of the 2nd fastq in paired-end reads. Column is not required if all fastqs are single-end '.' if the column is used, but this line is single-ended.	path/fastq2

The following columns are optional and used to populate the Read Group Information in the BAM file.

• RGID field is required if using any of these fields, the others are optional.

What is a Read Group?

• Groups reads together
• Used for recalibration
  • Each sequencing run should get a different ReadGroup

If you do not want the field for:

• any fastq, leave the column out of the header line
• a single line, use a '.'

Column Name	Description	Recommended Value
RGID	Read Group ID	Run ID
SAMPLE	Sample Name	Sample Name
LIBRARY	Library separate FASTQs for a sample that were prepped separately	if you don't know or it is all the same, use Sample Name
CENTER	Center Name	Name of the sequencing center producing the FASTQ
PLATFORM	Platform	CAPILLARY, LS454, ILLUMINA, SOLID, HELICOS, IONTORRENT, or PACBIO

MERGE_NAME	FASTQ1	FASTQ2	RGID	SAMPLE	LIBRARY	CENTER	PLATFORM 
Sample1	fastq/S1/F1_R1.fastq.gz	fastq/S1/F1_R2.fastq.gz	RGID1	SampleID1	Lib1	UM	ILLUMINA 
Sample1	fastq/S1/F2_R1.fastq.gz	fastq/S1/F2_R2.fastq.gz	RGID1a	SampleID1	Lib1	UM	ILLUMINA 
Sample2	fastq/S2/F1_R1.fastq.gz	fastq/S2/F1_R2.fastq.gz	RGID2	SampleID2	Lib2	UM	ILLUMINA 
Sample2	fastq/S2/F2.fastq.gz	.	RGID2	SampleID2	Lib2	UM	ILLUMINA 

The --fastq/FASTQ setting can be used to specify a prefix to the FASTQ1/FASTQ2 file paths that should be applied before using the files.

This file is specified either via the command line parameter --index_file or via the configuration file setting INDEX_FILE.

The command-line setting takes precedence over the configuration file setting.

GotCloud Configuration File

This file is created by you to configure GotCloud for your data.