combining fastq files

[mmmm]

Hi all,
we did a MiSeq run and we got low cluster density but got fastq files- then we repeated the run for the same samples and got better cluster density- Now how could I combine the fastq files/ sample so I can get better reads required for the analysis
for example I have the following fastq files for sample x

run1_X_read1.fastq
run1_X_read2.fastq

run2_X_read1.fastq
run2_X_read2.fastq

what is the command please used for this purpose?

[JackieBadger]

In unix

cat run1_X_read1.fastq run2_X_read1.fastq > R1.run1.run2.fastq

cat run2_X_read1.fastq run2_X_read1.fastq > R2.run1.run2.fastq

[mastal]

should be:

cat run1_X_read1.fastq run2_X_read1.fastq > R1.run1.run2.fastq

cat run1_X_read2.fastq run2_X_read2.fastq > R2.run1.run2.fastq

[GenoMax]

You may have checked this already but in case you have not then check the quality profiles for the two runs independently. You want both runs to be of good quality for combined downstream analysis.

[JackieBadger]

should be:

cat run1_X_read1.fastq run2_X_read1.fastq > R1.run1.run2.fastq

cat run1_X_read2.fastq run2_X_read2.fastq > R2.run1.run2.fastq

thanks for correcting

[grakocevic]

What is the analysis and the pipeline you plan on using? If it's anything that can make use of the read groups (like gatk bqsr) you might want to align separately and merge the bam files (if you even plan on aligning that is).

[mmmm]

combining fastq files

You may have checked this already but in case you have not then check the quality profiles for the two runs independently. You want both runs to be of good quality for combined downstream analysis.

have checked the reads quality on fastqc (before trimming low qulaity bases) and they look reasonable- do you think it is better to trimm the low quality bases (<20) first before combining fastq files or shall I combine them firstly them carry out the trimming after?

[GenoMax]

Either should be ok. You are only appending the contents of one file to another by doing "cat". You are not changing any individual sequence reads in any way by that operation.

[mmmm]

combining fastq files

Either should be ok. You are only appending the contents of one file to another by doing "cat". You are not changing any individual sequence reads in any way by that operation.

not sure if it is possible to combine a fastq file of 250bp read with a fastq file (of the same sample) but 300bp read??- is this applicable

[GenoMax]

not sure if it is possible to combine a fastq file of 250bp read with a fastq file (of the same sample) but 300bp read??- is this applicable

That is what you will be doing. Using "cat" you are appending contents of file 2 at end of file 1 to produce file 3. That way you are going to end up with file 3 that has reads with 2 different lengths in it.

If you want to do any absolute length based trimming then you should do that *before* you "cat" the files together.