Single Cell RNA-Seq : Should I trim Poly-T & Clontech sequences called as "Overrepresented sequences" by FastQC ?

Question

[gsr9999]

Dear Biostars Leaders,

I am a bioinformatician in our lab and I have received raw data(bcl files) on ~300 single cells RNA-Seq samples from a biologist in our lab. I ran bcl2fastq and then run FastQC tool on the fastq files. In FastQC output’s "Overrepresented sequences" category, it has classified half of my sample’s fastq files (~160) with WARNING annotation due to the presence of Poly-T tail sequence(s), and other Clontech sequences. I wonder if I need to trim these sequences before the alignment step ? I appreciate any advice . Other FastQC metrics like Basic Stats, Adapter Content, Per Base seq quality , etc have PASSED for all of my samples.

The Single Cell RNA-Seq was performed using TakaBio/Clontech's SMART-Seq v4 Ultra Low Input RNA Kit chemistry, and the samples were indexed with illumina's Nextera XT adapaters and Index sequences. I made sure to put Adapter Sequence and Sample indexes (i5 & i7) in the Sample Sheet file that was given as input for bcl2fastq. I used default settings of bcl2fastq and I believe it performed adapter trimming and demultiplexing automatically.

"GTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT" is the top Poly-T sequence Overrepresented that is present in at least 160 sample fastq files, and FastQC reports its Possible Source as "No Hit". I did a simple google search for this above mentioned Poly-T sequence, and other people seem to have observed the same. Should I ignore this ? or remove/trim this sequence :

http://bioinformatics.stemcells.cam.ac.uk/Files_for_transfer_LILA/Fernando/extras/SLX-9555.C89V9ANXX.s_1.r_1.fastqc.html

http://single-cell.clst.riken.jp/fastqc/GSE68981_QC/SRR2031413_2_fastqc.html

http://waxmanlabvm.bu.edu/waxmanlab/FASTQC/SRR/SRR6576929_1_fastqc.html

Other Poly-T tail sequences are reported by FastQC at lower frequencies. Other Overrepresented Sequences are annotated as “Clontech SMARTer…”, “Clontech Universal Primer Mix…” .

Thanks,

GSR

Here is the complete list of 29 "Overrepresented Sequences" reported on my fastq files. Please advice me on how to proceed :

"Overrepresented_Sequence"   \t   "Possible_Source"   \t   "Affected_Samples_Count"

GTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT  No Hit  160

TATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT  No Hit  19

GGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT  No Hit  11

GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG  No Hit  3

GTACATGGGAAGCAGTGGTATCAACGCAGAGTACATGGGAAGCAGTGGTA  Clontech SMARTer II A Oligonucleotide (100% over 25bp)  2

AAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAA  No Hit  1

ACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT  No Hit  1

CAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACA  No Hit  1

ATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT  No Hit  1

GTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTG  No Hit  1

TATCAACGCAGAGTACATGGGAAGCAGTGGTATCAACGCAGAGTACATGG  Clontech Universal Primer Mix Long (96% over 26bp)  1

ACGCAGAGTACATGGGAAGCAGTGGTATCAACGCAGAGTACATGGGAAGC  Clontech Universal Primer Mix Long (96% over 26bp)  1

TTGTTGTTGTTGTTGTTGTTGTTGTTGTTGTTGTTGTTGTTGTTGTTGTT  No Hit  1

GGTATCAACGCAGAGTACATGGGAAGCAGTGGTATCAACGCAGAGTACAT  Clontech SMARTer II A Oligonucleotide (100% over 25bp)  1

CTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCT  No Hit  1

TATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTA  No Hit  1

GAGTACATGGGAAGCAGTGGTATCAACGCAGAGTACATGGGAAGCAGTGG  Clontech Universal Primer Mix Long (96% over 26bp)  1

CCCATGTACTCTGCGTTGATACCACTGCTTCCCATGTACTCTGCGTTGAT  Clontech Universal Primer Mix Long (96% over 26bp)  1

GTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTA  No Hit  1

GTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAA  No Hit  1

GTATCAACGCAGAGTACATGGGAAGCAGTGGTATCAACGCAGAGTACATG  Clontech SMARTer II A Oligonucleotide (100% over 25bp)  1

TTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTT  No Hit  1

GAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGA  No Hit  1

GTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT  No Hit  1

TCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTC  No Hit  1

AGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAG  No Hit  1

TATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAA  No Hit  1

GTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAAA  No Hit  1

GGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTA  No Hit  1

[GenoMax]

While many aligners will handle these oddities you may want to scan/re-trim the data (even though bcl2fastq did it).

Did you look at Appendix C in Takara's manual for this kit which has instructions on what you need to do.